Helicobacter System And Uses Thereof

ABSTRACT

Helicobacter  based preparations comprising a pharmacologically active molecule of interest are disclosed, as well as methods of preparing and using said preparations. In particular,  Helicobacter pylori  vectors, vector plasmids and recombinant cells that include a sequence encoding a pharmacologically active molecule of interest useful in therapeutic treatments and/or vaccination against disease are provided. Delivery of the pharamacologically active molecules is provided at the mucosal surface, such as the gastric mucosa or nasal membranes, to provide effective and continuous delivery of a pharmacologically active agent. In some embodiments, the  Helicobacter  provides exposure of a desired molecule of interest though the surface of the  Helicobacter,  providing exposure of the antigen to the host at the gastric mucosa. Live  Helicobacter pylori  vaccines are also provided. Vectors and shuttle vector constructs of the  Helicobacter  are also disclosed.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application No.60/602,859, filed Aug. 20, 2004, and to Australian Patent ApplicationNo. 2004/904564, filed Aug. 13, 2004, and to U.S. Utility applicationSer. No. 11/202,249, filed Aug. 12, 2005, the text of all applicationsbeing specifically incorporated herein by reference.

BACKGROUND

1. Field of the Invention

The present invention relates generally to the field ofHelicobacter-based vector, plasmid vector and shuttle vector systems, asnovel Helicobacter constructs that include a non-Helicobacterpharmacologically active molecule of interest are provided. Theinvention also relates to the field of drug delivery, vaccines andtreatment methods, as compositions that provide for the administrationand/or delivery of non-Helicobacter molecules at the mucosa in vivo aredisclosed.

2. Related Art

Helicobacter pylori are a gram-negative spiral shaped bacterium foundalmost exclusively in the human gastric mucosa. The acidity of the humanstomach is an effective barrier to colonization by essentially allbacteria, with the exception of Helicobacter species.

H. pylori have been described as a causative agent of chronic infection.In particular, Helicobacter has been established to play a critical rolein peptic ulcer, gastric adenocarcinoma, and primary gastric lymphoma.

H. pylori have the unique ability to colonize and persist for decadeswithin the human gastric mucosa, despite development of a mucosalinflammatory and immune response. This characteristic renders H. pylorian interesting candidate for the delivery of selected agents though themucosa. However, this particular application has not found applicationin mucosal delivery systems in part owing to its involvement in avariety of diseases. A need continues to exist for a delivery systememploying these important organisms having a reduced risk of pathologyto the host.

The development of mucosal vaccines has also been hindered by the poorimmunogenicity of antigens delivered by conventional approaches becauseof natural barrier functions of the host that prevent access to themucosal compartment. Hence, a need continues to exist in the medicalarts for improved delivery mechanisms for pharmacologically activemolecules at the mucosal surface sufficient to elicit a useful andbeneficial immunogenic response. Such would provide an effective in vivodelivery system for pharmacological active agents, as well as aneffective method for immunization, i.e., antigen exposure at a mucosalsurface sufficient to elicit a general humoral and mucosal immuneresponse.

SUMMARY

The present invention is directed to overcoming the above-mentionedchallenges and others related to the use of Helicobacter and in thetreatment of disease. The present invention is exemplified in a numberof implementations and applications, some of which are summarized below.

In accordance with some aspects, compositions, methods and systems areprovided for preparing and using a Helicobacter-based constructcomprising a Helicobacter sequence having a promoter region and anon-Helicobacter sequence encoding a non-Helicobacter molecule ofinterest. This construct in some embodiments is described as a vector ora plasmid vector, wherein the promoter sequence is capable ofcontrolling the expression of the non-Helicobacter sequence encoding amolecule of interest.

Helicobacter Constructs:

In one aspect, the invention provides a composition comprising aHelicobacter construct, particularly a Helicobacter pylori nucleic acidconstruct. In some embodiments, the Helicobacter nucleotide sequence ofthe Helicobacter construct comprises a first Helicobacter sequence, Y1,a second Helicobacter sequence Y2, and a non-Helicobacter sequence Xencoding a non-Helicobacter molecule of interest. A schematic of oneembodiment of this construct appears in Formula 1:

The non-Helicobacter. nucleotide sequence of interest, “X”, may comprisea nucleic acid sequence that encodes a pharmacologically active and/orbiologically active molecule of interest. In some embodiments, thenon-Helicobacter sequence X is heterologous to the Helicobacter pylorispecies. A pharmacologically active molecule of interest or abiologically active molecule of interest may comprise an enzyme,protein, peptide, or other molecule. This molecule of interest may befurther described in some embodiments as capable of providing abeneficial and/or therapeutic effect to an animal delivered as anexpressed product from a recombinant Helicobacter containing theconstruct that is introduced into an animal. In some embodiments, themolecule of interest is ghrelin, amylin or an analog thereof.

In some embodiments, the construct comprises a first Helicobactersequence Y1 defined as a first portion of a native HopE gene sequence, asecond Helicobacter sequence Y2 defined as a second portion of a nativeHopE gene sequence, and a non-H. pylori nucleotide sequence of interest,“X”. By way of example, one such construct has a structure defined as:580 bp HopE—“X” bp—502 bp HopE. In some embodiments, “X” is a nucleicacid sequence comprising 60 nucleotide bases to 300 nucleotide bases, or69 nucleotide bases to 138 nucleotide bases.

In some embodiments, the Helicobacter construct comprises a construct asdepicted in FIG. 7.

In other embodiments, Helicobacter construct is further defined as anattenuated Helicobacter pylori construct.

The H. pylori nucleic acid construct may further comprise, in someembodiments, a promoter sequence, a secretory sequence and/or a reportergene sequence. In particular embodiments, a recombinant cell transformedwith the Helicobacter construct is provided. In some embodiments, theserecombinant cells are recombinant E. coli cells or H. pylori cells.

In some aspects, the Helicobacter-based vector and vector plasmidconstructs that contain the Helicobacter construct comprise apharmacologically active molecule of interest defined as an antigen,organic or inorganic molecule or substance, a pharmacologically activeagent, e.g. a therapeutic agent or prophylactic agent, such as a geneproduct or gene sequence (isolated nucleic acid). By way of example,such pharmacologically active molecules of interest may comprise animmunoregulatory agent, hormone, ligand, an enzyme, or an antisense RNA,a catalytic RNA, a protein, peptide or any other molecule. In someembodiments, the isolated nucleic acid molecule may be further describedas comprising cDNA, genomic DNA, RNA, or a hybrid molecule thereof. Inparticular embodiments, the nucleic acid is cDNA. In some embodiments,the vector construct is pTM103-8.

By way of example, a protein and/or peptide of interest may compriseghrelin, amylin, insulin, motilin, β-glucosidase, a chemical chaperone,or other molecule useful in the treatment and/or management of Gauchersdisease, cell wasting, human immunodeficiency disease (AIDS), appetitesuppression, preparations useful in the treatment of diabetes, etc.

Recombinant Cells:

In other aspects, a composition comprising a recombinant cell isprovided comprising cells transformed with the plasmid vectors and/orvectors that include a Helicobacter construct as described herein. Insome embodiments, the recombinant cell comprises a sequence encoding anon-Helicobacter pylori pharmacologically active molecule of interest.In other embodiments, the nucleic acid sequence encoding thenon-Helicobacter pylori pharmacologically active molecule of interestcomprises a secretory signal polypeptide. In some embodiments, therecombinant cell is capable of secreting an expressed productcorresponding to the non-Helicobacter molecule of interest at thesurface of the recombinant cell. In this manner, the expressed productof the molecule of interest may be delivered at or through the mucosalsurface of an animal, such as at the intestinal mucosa. In someembodiments the recombinant cell is a recombinant Helicobacter pyloricell such as a Helicobacter strain 26695 or B 128.

Pharmaceutical Preparations:

The present invention provides a variety of pharmaceutically acceptablepreparations formulated for delivery to a patient, such as, for example,delivery gastrically, orally, or intranasaly. In particular embodiments,the compositions are suitable for delivery at a mucosal surface. Inparticular embodiments, the composition is suitable for delivery to themucosal surface or lining. In some embodiments, the mucosal surface isthe gastric mucosal surface.

The various delivery forms of the compositions are readily prepared foruse in the practice of the present invention given the specific typesand ratios of specific Helicobacter, Helicobacter constructs and otherdelivery vehicles described herein, and those formulation techniquesknown to those in the formulary arts, such as are described inRemington's Pharmaceutical Sciences, 20^(th) edition, Mack PublishingCompany, which text is specifically incorporated herein by reference.

It is envisioned that the delivery system may be employed in animals,particularly primates, including humans, equines, bovines, ovines, androdents, fish and birds. It is also anticipated that the preparationsmay be used on both infants and adults, as well as parentally or foradministration to pregnant or lactating animals. The preparations andmethods may be further described as suitable for both male and femaleanimals.

Vaccines:

In some embodiments, the composition is further defined as a vaccine ina pharmacologically acceptable carrier solution. As part of a vaccine,the composition comprising the Helicobacter construct is introduced intoan animal in a manner such that the expressed product, i.e., themolecule of interest “X”, is capable of making contact with or at amucosal surface of an animal. By way of example, a mucosal surface of ananimal may include the gastric mucosa, the nasal mucosa, etc.

In some embodiments, the Helicobacter based vaccine comprises cellstransformed with a Helicobacter based construct, such as a plasmidvector as described herein. By way of example, the cells transformedwith the Helicobacter based plasmid vector may comprise E. coli cells orHelicobacter pylori cells. In some embodiments, the vaccine may befurther defined as a live attenuated vaccine. In particular embodiments,the composition will include an adjuvant. In some embodiments, thevaccine is capable of providing delivery of the non-Helicobactermolecule of interest at a mucosal surface.

Vaccination/Immunization:

In yet another aspect, a method is provided for vaccinating an animal.In some embodiments, the method comprises administering a compositioncomprising a vaccine comprising cells transformed with theHelicobacter-based vector and/or plasmid vectors as described herein. Inother embodiments, the method provides for the delivery of an effectiveamount of the pharmacologically active molecule of interest sufficientto eliminate or inhibit a disease or particular physiological and/orpathological condition in the animal, or sufficient to elicit an immuneresponse specific for the pharmacologically active molecule of interest.

By way of example, the non-Helicobacter molecule of interest that may beprovided to an animal in the vaccine preparations of the presentinvention may comprise a mammalian or non-mammalian protein, peptide,enzyme, hormone, or any combination of these. In particular embodiments,the molecule of interest is further defined as a pharmacologicallyactive molecule of interest that is a human pharmacologically activemolecule of interest. In some embodiments the pharmacologically activemolecule of interest is a human pathogen molecule/antigen, human proteinantigen, such as amylin or an analog or derivative thereof, ghrelin, oran analog or derivative thereof.

In particular embodiments, the vaccines of the present inventionprovides immunity and/or an enhancement of disease resistance to thehuman pathogen, Ebola virus, HIV virus, Marburg virus, influenza virus,and the like. Replication competent vaccines based on attenuatedrecombinant vesicular stomatitis virus vectors have been described byJones et al. (2005)⁴³ that include Ebola glycoprotein and Marburgglycoprotein. Hence, vaccine preparations containing constructs of theHelicobacter-based vector systems and plasmid vector systems describedherein, with these and other glycoproteins associated with humanpathogens, may also be provided according to the present invention.

In another aspect, a method of immunizing an animal is provided. In someembodiments, the method comprises providing a composition comprising theHelicobacter vaccine as described herein to an animal and administeringto the animal an effective amount of the composition sufficient toelicit an acceptable immune response in the animal. In some embodiments,the acceptable immune response is elicited upon the administration of atreatment regimen comprising one or more effective doses of thecomposition. These methods may be used in veterinary immunization aswell as in the immunization of humans.

The following nucleic acid and amino acid sequences are referencedthroughout the description of the present invention:

-   SEQ ID NO: 1—Nucleotide sequence of plasmid pHP1 (2796 nucleotides)    +ve strand.-   SEQ ID NO: 2—Nucleotide sequence of pHP1 (2796 nucleotides) −ve    strand.-   SEQ ID NO: 3—Nucleotide sequence of plasmid pHP3 (3444 nucleotides).-   SEQ ID NO: 4—Hepatitis C virus antigen (HCV) nucleotide Sequence    (580 nucleotides).-   SEQ ID NO: 5—Nucleotide sequence 135 bp (45 amino acids) immunogenic    coding sequence from the Hepatitis C virus (HCV) core antigen.-   SEQ ID NO: 6—Nucleotide sequence (1108 nucleotides) of the surface    exposed loop of the HopE gene (at nt504, aa position 168) of H.    pylori.-   SEQ ID NO: 7—Upsteam primer (29 nucleotides).-   SEQ ID NO: 8—Downstream Primer (28 nucleotides).-   SEQ ID NO: 9—Oligonucleotide Primer (15 nucleotides).

Nucleotide sequence of H. pylori insertion construct, HopE gene withnucleotide sequence of interest, “X”. (580 bp HopE—“X” bp—502 bp HopE).The nucleotide sequence of interest, “X”, may comprise a nucleic acidsequence that encodes a molecule of interest, such as a biologicallyvaluable molecule of interest. A biologically valuable molecule ofinterest may comprise an enzyme, protein, peptide, or other moleculethat is capable of providing a beneficial or therapeutic effect to ananimal as delivered as an expressed product though or at a mucosalsurface, such as the gastric mucosa. In some embodiments, “X” is anucleic acid sequence comprising 60 nucleotide bases to 150 nucleotidebases, or 69 nucleotide bases to 138 nucleotide bases.

Nucleotide sequence for fusion protein of HopE and p60, insertion of p60nucleic acid sequence (23 amino acids) at nucleic acid position 504(corresponding to amino acid (aa) position 168) in HopE sequence.

Nucleotide sequence for fusion protein of HopE and HCCA, insertion ofHCCA nucleic acid sequence (46 amino acids) at nucleic acid position 504(corresponding to amino acid (aa) position 168) in HopE nucleic acidsequence.

nucleic acid sequence for p60 (69 nucleotide bases).

nucleic acid sequence for HCCA (138 nucleotide bases).

BRIEF DESCRIPTION OF THE DRAWINGS

The invention will be described in conjunction with the accompanyingdrawings, in which:

FIG. 1, in accordance with one embodiment of the invention, illustratesthe vector constructs, pHPA1 (2.8 kb).

FIG. 2, in accordance with one embodiment of the invention, presents aschematic diagram of the plasmid construct pHP3 (3.4 kb).

FIG. 3 in accordance with one embodiment of the invention, illustratesthe vector construct, pTM103-8.

FIG. 4, in accordance with one embodiment of the invention, illustratesthe chemical structure of sulfasalazine (SSN).

FIG. 5, in accordance with one embodiment of the invention, illustratesa schematic using an ion exchange resin (Amberlite XE-96) conjugatedwith a dye (Azure-A).

FIG. 6, in accordance with one embodiment of the invention, illustratesthe predicted structure of HopE (SEQ ID NO: 55), showing the insertionsite for a sequence of interest, such as a nucleic acid sequenceencoding HCCA or p60 epitopes. Adapted from Bina and associates, whichreference is specifically incorporated herein by reference.

FIG. 7, in accordance with one embodiment of the invention, illustratesrecombinant DNA molecules produced using SOE PCR. DNA coding either theHCCA or p60 epitope was inserted into hopE at the position correspondingto aa 168 of HopE. To allow homologous recombination, and replacement ofthe genomic copy of hopE in H. pylori, sequences homologous to genomichopE were included flanking the insertion site for epitope coding DNA.

FIG. 8, in accordance with one embodiment of the invention, provides adiagrammatic representation of recombinant genes in H. pylori. Column A:Antigen (Ag) coding DNA was inserted into hopE at position correspondingto aa 168, within a region corresponding to a putative surface exposeloop; Column B: Antigen coding DNA was inserted at the cagA at the 5′terminus directly upstream of the cagA stop condon; Column C: B 128 vacAwas replaced DNA Coding the 26695 vacA promoter sequence, signalsequence (ss), mature (m) vacA, passenger domain and autotransporter(AT) domain. Antigen coding DNA was inserted directly upstream of thesignal sequence.

FIG. 9, in accordance with one embodiment of the invention, provides aWestern Blot analysis of H. pylori B128 (7,13) containing HCCA(B128:HCCA:hopE, Blot A. 1° antibody: α-HopE; 2° antibody; α-rabbit—APconjugate, Lane 1: marker; Lane 2: B128; Lane 3: B128:HCCA:hopE; Lane 4:B128:p60:hopE. Blot B: 1° antibody: α-HCCA; 2° antibody;α-mouse—AP-conjugate. Lane 1: marker; Lane 2: B128:HCCA; hopE; Lane 3:B128. Blot C. 1° antibody: α-p60; 2° antibody; α-mouse—Ap conjugate (2°antibody). Lane 1: marker; Lane 2: B128:p60:hopE; Lane 3: B128. Thewhite arrow indicates the band corresponding to either HopE or fusionprotein.

FIG. 10, in accordance with one embodiment of the invention, presence animmunofluorescence based microscopy analysis of H. pylori B128containing HCCA inserted into HopE (B128:HCCA:hopE). Upper Row: Phasecontrast microscopy; Lower Row: Fluorescence microscopy. Column A: H.pylori B128. 1° antibody: α-HopE; 2° antibody; α-rabbit Alexafluor 488(AF-488). Column B: H. pylori B128. 2° antibody; α-rabbit AF-488. ColumnC. H. pylori B128. 1° antibody: α-HCCA; 2° antibody: α-mouse AF-488.Column D: H. pylori B128:HCCA:hopE. 2° antibody: α-mouse AF-488. ColumnE. H. pylori B128:HCCA-hopE. 1° antibody: α-HCCA: 2° antibody: α-mouseAF-488.

FIG. 11, in accordance with one embodiment of the invention, presence awhole cell based ELISA analysis of recombinant H. pylori 26695 and B128(7.13). Column A: Recombinants 26695:HCCA:hopE or 26695:p60:hopE; ColumnB: Recombinants B128:HCCA:hopE B128:p60:hopE; Detection of p60 antigen:1° antibody: α-p60; 2° antibody: α-mouse-alkaline phosphatase (AP)conjugate; Detection of HCCA: 1° antibody: α-HCCA; 2° antibody:α-mouse-AP conjugate. Error bars: SEM (n=3).

DETAILED DESCRIPTION

The present invention is believed to be applicable to a variety ofdifferent types of bacterial and vaccine constructs that include aHelicobacter or Helicobacter-based vector system of delivery. It isadvantageous to define several terms before describing the invention.

While the present invention is not necessarily limited to suchapplications, various aspects of the invention may be appreciatedthrough a discussion of various examples using this context.

Description

Before describing the present invention in detail, it is to beunderstood that this invention is not limited to particularlyexemplified methods and may, of course, vary. It is also to beunderstood that the terminology used herein is for the purpose ofdescribing particular embodiments of the invention only, and is notintended to be limiting which will be limited only by the appendedclaims.

All publications, patents and patent applications cited herein, whethersupra or infra, are hereby incorporated by reference in their entirety.However, publications mentioned herein are cited for the purpose ofdescribing and disclosing the protocols, reagents and vectors which arereported in the publications and which might be used in connection withthe invention. Nothing herein is to be construed as an admission thatthe invention is not entitled to antedate such disclosure by virtue ofprior invention.

Furthermore, the practice of the present invention employs, unlessotherwise indicated, conventional immunological and molecular biologicaltechniques and pharmacology within the skill of the art. Such techniquesare well known to the skilled worker, and are explained fully in theliterature. See, e.g., Coligan et al., (Current Protocols in ProteinScience (1999) Volume I and II (John Wiley & Sons Inc.); Sambrook etal., (Molecular Cloning: A Laboratory Manual, 2nd & 3rd Editions. ColdSpring Harbor Laboratory press (1989) (2001); and Bailey, S F. andOllis, D. F., Biochemical Engineering, Fundamentals. McGraw-Hill BookCompany, NY, 1986.

It must be noted that as used herein and in the appended claims, thesingular forms “a,” “an,” and “the” include plural reference unless thecontext clearly dictates otherwise. Thus, for example, a reference to “anucleic acid” includes a plurality of such nucleic acids, and areference to “an isolated peptide” is a reference to one or morepeptides, and so forth.

Unless defined otherwise, all technical and scientific terms used hereinhave the same meanings as commonly understood by one of ordinary skillin the art to which this invention belongs. Although any materials andmethods similar or equivalent to those described herein can be used topractice the present invention, the preferred materials and methods arenow described.

Delivery of therapeutic compositions and nucleic acids to specifictarget sites within the animal body is an ongoing challenge faced by thedrug development industry. The present inventor has developed aHelicobacter-based bacterial delivery system capable of carrying vectorsencoding biologically active agents, wherein these agents are expressedon the surface of the bacterium or secreted there from. In oneembodiment, the bacterium is a species of Helicobacter, H. pylori. Insome embodiments, the strain of H. pylori can be any strain known in thefield. In some embodiments, the H. pylori strain is a non-pathogenicstrain such as genomic strain 26695. Another strain that may be used isH. pylori strain B128, particularly variant 7.13.

In another embodiment, a bacterium, other than Helicobacter, is utilizedwherein the bacterium has been genetically altered such that it hasHelicobacter or H. pylori features including the ability to chronicallycolonize the gastric mucosa or other areas of gastrointestinal tract,urinary tract, bronchial epithelium or other mucosal surface, withoutsignificant toxicity to the host.

In one embodiment, the H. pylori have been manipulated so that some ofthe pathogenic features have been removed and/or attenuated. Forexample, the vacuolating cytotoxin and the cag pathogenicity islandgenes can be removed so that the H. pylori are less pathogenic.Attenuating mutations can be introduced into Helicobacter usingnon-specific mutagenesis either chemically, usingN-methyl-N-nitro-N-nitrosoquanidine, or using recombinant DNAtechnologies.

The skilled person will appreciate that the methods of the presentinvention could be used to deliver biologically active agents. Examplesof suitable agents include ones which are capable of functioning locallyor systemically, e.g., an agent capable of exerting endocrine activitiesaffecting local or whole-body metabolism and/or an agent which iscapable of regulating the activities of cells belonging to theimmuno/hematopoeitic system and or an agent which is capable ofaffecting the viability, growth and differentiation of a variety ofnormal or neoplastic cells in the body or affecting the immuneregulation or induction of acute phase inflammatory responses to injuryand infection and/or an agent which is capable of enhancing or inducingresistance to infection of cells and tissues mediated by chemokinesacting on their target cell receptors, or the proliferation ofepithelial cells or the promotion of wound healing and/or an agent whichmodulates the expression or production of substances by cells in thebody.

Specific examples of such biologically active agents include insulin,growth hormone, prolactin, calcitonin, luteinizing hormone, parathyroidhormone, somatostatin, thyroid stimulating hormone, vasoactiveintestinal polypeptide, a structural group 1 cytokine adopting anantiparallel 4 α helical bundle structure such as IL-2, IL-3, IL-4,IL-5, IL-6, IL-7, IL-9, IL-10, IL-11, IL-12, IL-13, CM-CSF, M-CSF, SCF,IFN-γ, EPO, G-CSF, LIF, OSM, CNTF, GH, PRL or IFN α/β, a structuralgroup 2 cytokine which are often cell-surface associated, form symmetrichomotrimers and the subunits take up the conformation of β-jelly rolldescribed for certain viral coat proteins such as the tumor necrosisfactor (TNF) family of cytokines, e.g. TNF α, TNF β, CD40, CD27 or FASligands, the IL-1 family of cytokines, the fibroblast growth factorfamily, the platelet derived growth factors, transforming growth factorβ and nerve growth factors, a structural group 3 cytokine comprisingshort chain α/β molecules, which are produced as large transmembranepre-cursor molecules which each contain at least one EGF domain in theextracellular region, e.g., the epidermal growth factor family ofcytokines, the chemokines characterized by their possession of aminoacid sequences grouped around conserved cysteine residues (the C—C orC—X—C chemokine subgroups) or the insulin related cytokines, astructural group 4 cytokine which exhibit mosaic structures such as theheregulins or neuregulins composed of different domains, e.g., EGF,immunoglobulin-like and kringle domains.

Alternatively, the biologically active agent can be a receptor orantagonist for biologically active agent as defined above.

In some embodiments, the H. pylori-based vector and/or vector plasmidconstruct is employed to create a transformed cell (such as an E. colicell or Helicobacter cell) that permits the expression and secretion ofa non-Helicobacter pharmacologically active molecule of interest at themucosal membrane of a host to which the transformed cell preparation isadministered. The isolated nucleic acid molecule contained within thetransformed cell (or vector) may comprise one or more nucleic acidconstructs in which nucleic acid encoding the pharmacologically activemolecule of interest is under control of H. pylori regulatory sequences.

Suitable vectors and shuttle vector sequences can be chosen orconstructed to contain appropriate regulatory sequences, includingpromoter sequences, terminator fragments, enhancer sequences, markergenes and other sequences as appropriate. Vectors may be plasmids,viral, e.g. phage, or phagemid, as appropriate. For further details, forexample, see Sambrook et al., supra. Many techniques and protocols areknown for the manipulation of nucleic acid, for example in preparationof nucleic acid constructs, mutagenesis, sequencing, introduction of DNAinto cells and gene expression, and analysis of proteins, as describedin detail in Short Protocols in Molecular Biology, Second Edition,Ausubel et al. eds., John Wiley & Sons, 1992. The disclosures ofSambrook et al. supra and Ausubel et al. are incorporated specificallyherein by reference.

In some embodiments, the coding sequence(s) for the pharmacologicallyactive molecules of interest is contained in an operon, i.e., a nucleicacid construct for multi-cistronic expression. In an operon,transcription from the promoter results in a mRNA which comprises morethan one coding sequence, each with its own suitably positioned ribosomebinding site upstream. Thus, more than one agent (pharmacologicallyactive molecule of interest) can be translated from a single mRNA. Useof an operon enables expression of the pharmacologically active moleculeof interest to be coordinated.

A nucleic acid construct or vector comprising a coding sequence for apharmacologically active molecule of interest is preferably under thecontrol of a promoter for expression in H. pylori.

In one embodiment, the promoter employed in accordance with the presentinvention is expressed constitutively in H. pylori. Use of aconstitutive promoter avoids the need to supply an inducer or otherregulatory signal for expression to take place. Preferably, the promoterdirects expression at a level at which the H. pylori host cell remainsviable, i.e., retains some metabolic activity, even if growth may bereduced. Advantageously then, such expression may be at a low level. Forexample, where the expression product accumulates intracellularly, thelevel of expression may lead to accumulation of the expression productat less than about 10% of cellular protein, preferably about or lessthan about 5%, for example about 1-3%.

In some embodiments, a method is provided comprising delivering amessenger nucleic acid sequence, such as an mRNA sequence, correspondingto a nucleic acid sequence encoding a molecule of interest to an animal.By way of example, such a messenger nucleic acid sequence (mRNA)corresponding to a therapeutic peptide, protein, hormone or pro-hormonemay be prepared so as to provide a peptide, protein, or hormone to theanimal upon expression of that messenger nucleic acid sequence in theanimal. For example, such a hormone may be insulin, and such apro-hormone may be pro-insulin. Thus, it is envisioned that the presentinvention has application as a gene therapy method for the treatment ofhuman disease, such as for the treatment of diabetes.

The promoter may be homologous to the H. pylori strain employed, i.e.one found in that strain of H. pylori in nature. In some embodiments,the promoter is an arabinose inducible promoter. Other promoters includeFlaB sigma 54 promoter (Josenhans et at., 1998, FEMS Microbiol Lett,161(2): 263-73), T7 promoter, and nir B promoter of Salmonella(Chatfield et al., 1992, Biotechnology, 10(8): 888-92).

In another embodiment the promoter is inducible. Inducible promotersthat may be used with clinical grade vectors include, but are notlimited to, an inducible promoter as described in U.S. Pat. No.6,242,194 issued to Kullen et at., a lactose inducible promoter such, asthat used in E. coli plasmids (e.g., pBluescript™ from Stratagene) orthe endogenous lactose promoter in Lactobacillus, and promoters inducedduring anaerobic growth, such as the promoter for alcohol dehydrogenase(adhE), as described in Aristarkhov et at., (1999) J. Bacteriology,178(14), 4327-4332).

In one embodiment, the constructs of the present invention also includea toxic gene. These toxic genes are preferably under the control of aninducible promoter so that, on completion of treatment, the Helicobactercan be readily eliminated by inducing the expression of the toxic gene.Non-limiting examples of toxic genes include bacterial autolysins underthe control of an inducible promoter. The autolysing gene may then betriggered at the appropriate time and place in the gastrointestinaltract through the use of one or more of the inducible promoters asdescribed herein.

In some embodiments, the engineered Helicobacter vector and plasmidvector constructs are sensitive to oxygen. This oxygen sensitivity isanother method for limiting dissemination of the clinical grade vectorsof the present invention. The environment of the human gut is very lowin oxygen, suitable for growth of anaerobic and microacrophulicmicroorganisms, including Helicobacter. Thus, an efficient means ofeliminating Helicobacter, once they have exited the human body upondischarge of intestinal waste into the oxygen-rich outside environment,is to engineer genes into the transformed microorganisms that conferoxygen sensitivity.

The nucleic acid construct or constructs of the present invention mayalso comprise a secretory signal sequence. Thus, in some embodiments,the nucleic acid encoding the pharmacologically active molecule ofinterest (for example, a non-Helicobacter polypeptide) may provide forsecretion of the molecule at a cell membrane by appropriately coupling anucleic acid sequence encoding a secretory signal sequence to thenucleic acid sequence encoding the molecule (polypeptide). The abilityof Helicobacter harboring the nucleic acid to secrete the polypeptidemay be tested in vitro in culture conditions, which maintain viabilityof the Helicobacter.

Suitable secretory signal sequences include any of those with activityin Gram negative organisms such as Escherichia, Klebsiella andSalmonella. Secretory signal sequences include the Staphylokinase enzymesecreted by some strains of Staphylococcus, which is known to functionin both Gram-positive and Gram-negative hosts (see “Gene ExpressionUsing Bacillus”, Rapoport (1990), Current Opinions in Biotechnology,1:21-27).

Other secretory signal sequences that can be used include, for example,the β-lactamase gene (Talmadge et at., 1980, Proc. Natl. Acad Sci. USA77:3369-3373) or the enteroinvasive E. coli hemolysin A (hlyA) (Su etat., 1992, Microbial Pathogen, 13:465-476). An illustrative list ofsecretory signal sequences is presented in Pugsley, 1988, Proteinsecretion across the outer membrane of gram-negative bacteria. In:Protein Transfer and Organelle Biogenesis, R. C. Dand and P. W. Robbins(eds). Academic Press, Inc., San Diego, pp 607-652.

Selectable markers provide researchers and technicians a convenientmeans for distinguishing transformed microorganisms from non-transformedones in a mixed population. One means of identifying transformedorganism is to incorporate a selectable marker nucleic acid sequenceinto the plasmid containing the gene of interest. The selectable markersequence is generally inserted downstream of the gene of interest and isdriven off the same promoter. As a result, cells successfullytransformed with the gene of interest will also be transformed with theselectable marker nucleic acid sequence. When antibiotic resistance isused as the selectable marker, only transformed cells will surviveand/or grow in media containing the antibiotic.

Thus, antibiotic resistance is a convenient and much used phenotype whendeveloping transformants. However; vectors having antibiotic resistantgenes as selective markers are capable of horizontal gene transfer thatcan endow other organisms with antibiotic-resistant phenotypes. Thisrisk is especially acute when Helicobacter is used as part of atherapeutic vector.

In order to use Helicobacter as a gene delivery system to animals, thepresent disclosure presents, in some embodiments, a clinical gradevector system that does not use an antibiotic selection marker. One ofthe alternatives to using antibiotic resistance genes provided by thepresent delivery systems includes clinical grade vectors havingchromosomal deletions or lethal mutations in a “house-keeping” gene.Next, a functional analogous house-keeping gene is inserted into aplasmid encoding for the pharmacologically active molecule of interest.Consequently, the house-keeping gene becomes the selectable markerallowing for the rapid isolation and identification of transformants.

Examples of “house keeping genes” include genes that encode for anynumber of metabolic regulators and/or enzymes including, but not limitedto kinases, proteases, synthetases, dehydrogenases and others. Anotheralternative to antibiotic resistance genes provided by the presentinvention includes clinical grade vectors having reporter genesincorporated into the plasmid containing the gene encoding for thepharmacologically active molecule of interest. Other examples ofreporter genes used in accordance with the teachings of the presentinvention include Green Fluorescent Protein (GFP), β-galactosidase andamylase.

In one embodiment, the pharmacologically active molecule of interest hascytokine activity. Cytokines are discussed in The Cytokine Facts Rook,Callard and Gearing (1994), Academic Press. Preferred molecules, such aspolypeptides with cytokine activity are interleukins, includingInterleukin-2 (IL-2) and Interleukin 6 (IL-6).

In some embodiments, the Helicobacter vector and plasmid vector systemscomprise a nucleic acid construct as described above that is introducedinto a Helicobacter or other suitable host cell, to provide transformedcells. Thus, a further aspect provides a method comprising introducingnucleic acid as disclosed into a non-pathogenic Helicobacter.Transformation of a culture of host cells, such as Helicobacter, mayemploy any available technique. For H. pylori cells, suitable techniquesmay include calcium chloride transformation, electroporation andtransfection using bacteriophage.

The introduction of the plasmid vector into a Helicobacter cell may befollowed by causing or allowing expression from the nucleic acid, e.g.,by culturing H. pylori under conditions suitable for expression of thegene. Growing the Helicobacter in culture under conditions forexpression of the pharmacologically active molecule of interest may beemployed to verify that the Helicobacter contain the encoding nucleicacid and is able to produce the encoded molecule.

In a further aspect, the present invention provides a method ofdelivering a therapeutic or prophylactic dose of a biologically activeagent in vivo, the method comprising administering to a subject aneffective amount of the non-pathogenic preparation of the H. pyloricompositions and vaccines of the present invention.

It will be appreciated that the methods of the present invention and theuse of a non-invasive or non-pathogenic Helicobacter as described hereinprovide a wide range of therapeutic methods which would enable theskilled person to manipulate, for instance, the immune response of asubject. Thus, in one aspect, a method of regulating the survival,growth, differentiation, effector functions or susceptibility toinfection of cells or tissues is provided which comprises administeringto a subject a non-invasive or non-pathogenic Helicobacter as definedherein.

In another aspect, a method of boosting an immune response against tumorcells or an infection colonizing a mucosal surface or adjacent ordistant tissue is provided which comprises administering to a subject anon-invasive or non-pathogenic Helicobacter as defined herein.

In yet another aspect, a method of modulating the type of immuneresponse (antibody versus cell-mediated) against a pathogenic infectiousagent is provided which comprises administering to a subject anon-invasive or non-pathogenic Helicobacter as defined herein.

In another aspect, a method of modulating the infiltration of normaltissues with inflammatory or tumor cells is provided which comprisesadministering to a subject a non-invasive or non-pathogenic Helicobacteras defined herein.

In some aspects, a method of controlling the rate of growth, rate ofinvasion or survival of tumor cells is provided which comprisesadministering to a subject a non-invasive or non-pathogenic Helicobacteras defined herein.

In yet another aspect, a method of inducing apoptosis in tumor cells isprovided which comprises administering to a subject a non-invasive ornon-pathogenic Helicobacter as defined herein.

Other aspects provide for a method of down-regulating an immune responsewhich comprises administering to a subject a non-invasive ornon-pathogenic bacterium which expresses a pharmacologically activemolecule of interest as defined herein.

In another aspect, a method of treating an allergic autoimmune or otherimmune dysregulative disease state is provided which comprisesadministering to a subject a non-invasive or non-pathogenic Helicobacterwhich expresses a pharmacologically active molecule of interest.

The subject can be any primate, equine, bovine, porcine, ovine, rodent,fish, or bird. In one embodiment, the subject is human. Administrationmay conveniently be nasal or oral.

In a therapeutic context, i.e., where the pharmacologically activemolecule of interest is a biologically active agent that provides abeneficial effect to the subject, the amount of the agent and/ortreatment regimen will preferably be provided in a “therapeuticallyeffective amount”, this being sufficient to show benefit to a subject.Such benefit may be at least amelioration or a reduction in the severityor occurrence of at least one symptom. In a prophylactic context, theamount may be sufficient to reduce the deleterious effect on the subjectof a subsequent pathogenic challenge, for instance by enhancing theimmune response. The actual amount administered, and rate andtime-course of administration will depend on the aim of theadministration, e.g., the biological effect sought in view of the natureand severity of the challenge, and is the subject of routineoptimization. Prescription of treatment, including prophylacticvaccination, for example, decisions on dosage etc, is within theresponsibility of general practitioners and other medical doctors.

A composition comprising Helicobacter may be administered in accordancewith the present invention alone or in combination with othertreatments, either simultaneously or sequentially.

The present invention also provides a pharmaceutical compositioncomprising a Helicobacter as disclosed. Such a pharmaceuticalcomposition is in one embodiment preferably suitable for application toa mucosal membrane.

Pharmaceutical compositions according to the present invention, and foruse, may comprise, in addition to the Helicobacter, a pharmaceuticallyacceptable excipient, carrier, buffer, stabilizer or other materialswell known to those skilled in the art. Such materials should benon-toxic and should not interfere with the efficacy of thepharmacologically active molecule of interest. The nature of the carrieror other material may depend on the route of administration. For oraladministration a parenterally acceptable aqueous solution may beemployed which is pyrogen-free and has suitable pH, isotonicity andstability. Those of relevant skill in the art are well able to preparesuitable solutions. Preservatives, stabilizers, buffers, antioxidantsand/or other additives may be included, as required. As discussed, apharmaceutical comprising a Helicobacter for administration inaccordance with the present invention may comprise one or more nutrientsubstances, e.g., an energy source such as glucose, amino acids and soon.

In another aspect, a method of manufacture of a pharmaceuticalformulations provided comprising formulating Helicobacter as disclosedwith a suitable carrier medium suitable for administration to anindividual. In one embodiment, the pharmaceutical is suitable forapplication to a mucosal membrane of an individual.

In yet another aspect, a non-pathogenic Helicobacter expressing aheterologous pharmacologically active molecule of interest forpharmaceutical use is provided, e.g., for use in a method of treatmentof the human or animal body by surgery or therapy, including prophylaxis(“vaccination”).

In one embodiment the method can be used to treat, prevent or palliate adisease such as cancer. The methods and delivery system can also be usedto treat or prevent a disease or condition of the immune/hematopoieticsystem, a disease or condition of the reproductive system, a disease orcondition of the musculoskeletal system, a disease or condition of thecardiovascular system, a disease or condition described as mixed fetal,a disease or condition of the excretory system, a disease or conditionof the neural/sensory system, a disease or condition of the endocrinesystem, a disease or condition of the respiratory system, a disease orcondition of the digestive system and a disease or condition associatedwith connective/epithelial tissue or disease or condition caused bybacterial, viral or parasitic infection.

In another embodiment, the Helicobacter delivery system described hereinis capable of concomitant or sequential delivery of a number ofdifferent nucleic acid molecules, which encode products capable oftreating a number of conditions or diseases described herein. Moreover,preferred delivery systems would also deliver compositions capable ofproducing additional desirable physiological effects such as appetitesuppression or enhancement.

An example of suicide system in H. pylori has been described by Panthelet al. 2003 (Infection & Immunity, 71: 109-116). This system introducesa plasmid into H. pylori which contains the PhiX174 lysis gene E. Toeradicate the strain, incubation at 42° C. for 5 hours was used. In vivothis would mean that the animal would consume a drink at 45-50° C. toraise the temperature of the gastric environment above 42° C.

A second example is the L-Dap selection system, commonly used to allowsurvival of bacterial mutants on supplemented plates (see, for example,Kirata et al. 1997 (Infection & Immunity, 65: 4158-4164)). In thissystem the animal subject must supplement their diet with a missingsubstrate i.e., diamino-pimelic-acid (DAP), in order for the DapEdeficient H. pylori mutant to survive. In order to eradicate themutants, DAP consumption is ceased.

A third possible system relates to metronidazole sensitivity of H.pylori because of its rdxA gene. Excessive replication of the rdxA geneis harmful to mammalian cells and E. coli. However, duplication may betolerated by the bacterium. Therefore a Helicobacter species of thepresent invention can be engineered to contain two copies of rdxA whichprevents the normal mutation-dependant rdxA loss. The introduction of atleast two functional rdxA genes into the Helicobacter genome will resultin a Helicobacter strain that is permanently sensitive to metronidazole.Jeong et al. 2000 (J. Bacteriol., 182: 5082-5090) showed that thenitroreductase produced by a functional rdxA gene converts metronidazolefrom a prodrug to a bactericidal compound. The mode of action of theactive compound is to cause DNA breaks of the Helicobacter genome.

Throughout the specification, unless the context requires otherwise, theword “comprise” or variations such as “comprises” or “comprising”, willbe understood to imply the inclusion of a stated integer or group ofintegers but not the exclusion of any other integer or group ofintegers.

Definitions:

Prior to setting forth the invention, it may be helpful to anunderstanding thereof to set forth definitions of certain terms thatwill be used hereinafter.

The term “a” and “the” as used in the present descriptive is intended toinclude both one (the singular) and more than one (plural).

The phrase, “effective level” refers to the level of the desiredactivity of the pharmacologically active molecule of interest and notnecessarily limited to the number of molecules. For example, theeffective level of amylin (as an exemplary pharmacologically activemolecule of interest) may be decreased to stimulate ghrelin secretion byusing amylin antagonists, without a necessary concomitant decrease inthe amount of free amylin present in a subject.

An “antibiotic resistance gene” as defined herein includes heterologousnucleic acid sequences purposely provided to a vector and used as aselection system. The term “antibiotic resistance gene” does not includeother mechanisms or genes that impart antibiotic resistance to naturallyoccurring micro-flora organisms.

The term “attenuated” as used herein for example to describe a bacterialstrain, particularly an E. coli or a Helicobacter strain such asHelicobacter pylori, is defined as a strain that is less virulent and/ortoxic (invasive) that a native, wild type bacterial strain.

The term “biologically active” as used herein refers to ability toperform a biological function and with reference to a polypeptideimplies that the polypeptide adopts a stable conformation (“foldedform”) which is the same or closely analogous to its nativeconformation. When folded correctly or substantially correctly, forexample with formation of proper folded units' α-helices, β-sheets,domains, disulphide bridges etc., a polypeptide should have the abilityto perform its natural function. Generally, the unit of function in apolypeptide is a domain.

“Clinical grade vector” as used herein means a plasmid or otherexpression vector that is capable of being expressed in Helicobacter ora non-pathogenic bacterium engineered to have features of Helicobacter.The clinical grade vectors of the present invention do not useantibiotic resistance markers for selection and/or have been modified toprevent replication outside the host e.g., such as a suicide vector.

“Detectable immune response” as used herein is either an antibody(humoral) or cytotoxic (cellular) response formed in an animal inresponse to an antigen that can be measured using routine laboratorymethods including, but not limited to enzyme-linked immunosorbent assays(ELISA), radio-immune assays (RIA), Enzyme-linked ImmunoSPOT (ELISPOT),immunofluorescence assays (IFA), complement fixation assays (CF),Western Blot (WB) or an equivalent thereto.

“Gene of interest” as used herein refers to any nucleic acid sequenceencoding for a pharmacologically active molecule of interest, such as,polypeptide or protein, whose expression is desired. The nucleic acidsequence may or may not include the promoter or other regulatorycomponents. The vectors and plasmid vectors also include constructscapable of producing anti-sense RNA.

“Gene therapy” as used herein is defined as the delivery of a gene ofinterest to an animal in need thereof using a recombinant vector. Thegene of interest can be a transgene encoding for a therapeutic orprophylactic protein or polypeptide including, but not limited tocytokines, anti-inflammatories, anti-proliferatives, antibiotics,metabolic inhibitors/activators and immunologically active antigens andfragments thereof. Furthermore, “gene therapy” as used herein alsoincludes gene replacement technologies directed at both inherited andnon-inherited disorders.

The term Helicobacter includes all bacteria of the genus Helicobacterincluding H. pylori and Helicobacter mustelae. The term also includesbacteria that have similar biology to H. pylori in that they are capableof residing on the gastric mucosa of primates and/or capable ofestablishing a chronic, but isolated infection of the mucosa. The termalso encompasses bacteria that have been modified so that the bacteriumhas H. pylori features, such as the ability to reside on the gastricmucosa.

A “heterologous” polypeptide is a peptide that is not native or that hasbeen mutated from the native form as it existed in Helicobacter, i.e.,not expressed by Helicobacter in nature or prior to introduction intoHelicobacter, or an ancestor thereof.

“Host” as used herein defines the intended recipient of a therapeuticcomposition of the present invention. Host includes all animals.Specifically, hosts include, but are not limited to, primates (includingman), bovine, equine, canine, feline, porcine, ovine, rabbits, rodents,birds and fish.

“Immunologically inert” as used herein shall mean any substance,including microorganisms such as microflora that does not provoke asignificant immune response in its host. Examples of immunologicallyinert materials as used herein include stainless steel, biocompatiblepolymers such as poly-L-lactide, medical grade plastics and themicroflora organisms of the present invention.

An “insertion construct”, as used herein, shall mean a nucleic acidconstruct that comprises a portion of Helicobacter pylori nucleic acidsequence, such as a portion of a nucleic acid sequence that encodes theHopE gene, and a non-Helicobacter nucleic acid sequence that encodes amolecule of interest.

An “isolated nucleic acid” is a nucleic acid sequence that is notidentical to any naturally occurring nucleic acid or any fragment of anaturally occurring genomic nucleic acid sequence spanning more thanthree separate genes. The term therefore covers, for example, (a) a DNAmolecule which has the sequence of part of a naturally occurring genomicDNA molecule but is not flanked by both of the coding sequences thatflank that part of the molecule in the genome of the organism in whichit naturally occurs; (b) a nucleic acid incorporated into a vector orinto the genomic DNA of a prokaryote or eukaryote in a manner such thatthe resulting molecule is not identical to any naturally occurringvector or genomic DNA; (c) a separate molecule such as a cDNA, a genomicfragment, a fragment produced by polymerase chain reaction (PCR), or arestriction fragment; and (d) a recombinant nucleotide sequence that ispart of a hybrid gene, i.e., a gene encoding a fusion protein.

“Percent identity” (homology) of two amino acid sequences or of twonucleic acids is determined using the algorithm of Karlin and Altschul,1990, Proc. Natl. Acad. Sci. USA. 87:2264-2268, 1990, modified as inKarlin and Altschul (Proc. Natl. Acad. Sci. USA 90:5873-5877, 1993).Such an algorithm is incorporated into the NBLAST and XBLAST programs ofAltschul et al. (J. Mol. Biol. 215:403-410, 1990). BLAST nucleotidesearches are performed with the NBLAST program, score=100,wordlength=12, to obtain nucleotide sequences homologous to a nucleicacid molecule of the invention. BLAST protein searches are performedwith the XBLAST program, score=50, wordlength=3, to obtain amino acidsequences homologous to a reference polypeptide (e.g., SEQ ID NO: 2). Toobtain gapped alignments for comparison purposes, Gapped BLAST isutilized as described in Altschul et al. (Nucleic Acids Res.25:3389-3402, 1997). When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and NBLAST)are used.

A “pharmacologically active” molecule, as used in the description of thepresent invention, is defined as a molecule, such as a peptide, protein,nucleic acid, or other organic or inorganic substance that is capable ofeliciting a pharmacologically detectable activity or response in a cell,such as in a cell culture, or in a chemical or biochemical reactionmedia or assay, or in an animal. The pharmacologically active moleculesof interest of the present invention may include, for example,biologically active molecules as described herein.

A “molecule of interest” as used in the description of the presentinvention, is defined as a protein, peptide, enzyme, or other moleculethat when provided to a cell or animal, provides a protein, peptide,enzyme or other molecule that is capable of correcting and/or treating apathology, deficiency or other condition deemed appropriate, such as inthe treatment of a disease.

The term “reporter gene” as used herein is a nucleic acid sequenceincorporated into (or adjacent to) the heterologous nucleic acidsequence encoding a pharmacologically active molecule of interest thatprovides the transformed vector expressing the molecule of interest anidentifiable phenotype. Non-limiting examples of reporter genes includeGFP, β-galactosidase, amylase and CAT.

“Screening marker” as used herein refers to an identifyingcharacteristic (phenotype) provided to a transformed vector made inaccordance with the teachings of the present invention. In oneembodiment of the present invention, the screening marker is a reportergene.

“Selectable marker,” “selectable gene,” “reporter gene” and “reportermarker” (referred to hereinafter as a “selectable marker”) as usedherein refer to nucleic acid sequences encoding for phenotypic traitsthat permit the rapid identification and isolation of a transformedbacterial vector. Generally, bacterial vectors deemed “clinical grade”and made in accordance with the teachings of the present invention arethose vectors having selectable markers that do not encode forantibiotic resistance.

A “significant immune response” is any immune response that wouldprovide immunity (i.e., invoke the production of specific antibody) inan animal against a given antigenic molecule or immunogen.

A “therapeutically effective amount” of a pharmacologically activemolecule of interest or combination of said molecules as describedherein is understood to comprise an amount effective to elicit thedesired response but insufficient to cause a toxic reaction. A desiredresponse, for example, may constitute the formation of a sufficientand/or acceptable detectable antibody titer level in a blood sample. Thedosage and duration of treatment of the preparation to be administeredto a subject will be determined by the health professional attending thesubject in need of treatment, and will consider the age, sex, weight,extent of existing diseased state and/or tissue damage of the subject,and specific formulation of Helicobacter and the gene of interestproduct being used as the treatment for the subject.

A “transgene” as used herein refers to a gene that is inserted, usingcDNA technology, into a cell in a manner that ensures its function,replication and transmission as a normal gene.

A “transforming nucleic acid sequence” as used herein means a plasmid,or other expression cassette containing a nucleic acid sequence encodinga pharmacologically active molecule of interest. In some embodiments ofthe present invention, the nucleic acid sequence can encode for one ormore therapeutic agents. “Transforming nucleic acid sequence” can alsobe used to mean a “transgene” in accordance with certain embodiments ofthe present invention. In another embodiment of the present inventionthe transforming nucleic acid sequence includes nucleic acid sequenceencoding for a promoter and/or other regulatory elements.

The term “cancer” as used herein refers to neoplastic diseases eg.,leukemia, cancers and “hyperproliferative disorders”). The neoplasm maybe located in a tissue selected from the group consisting of: colon,abdomen, bone, breast, digestive system, liver, pancreas, prostate,peritoneum, lung, blood (e.g., leukemia), endocrine glands (adrenal,parathyroid, pituitary, testicles, ovary, thymus, thyroid), uterus, eye,head and neck, nervous (central and peripheral), lymphatic system,pelvic, skin, soft tissue, spleen, thoracic, and urogenital.

In one embodiment the term “cancer” also encompasses pre-neoplasticconditions selected from the group consisting of hyperplasia (e.g.,endometrial hyperplasia), metaplasia (eg, connective tissue metaplasia)and/or dysplasia (e.g., cervical dysplasia, and bronchopulmonarydysplasia).

In another embodiment, the term “cancer” also encompasses benigndysproliferative disorder selected from the group consisting of: benigntumors, fibrocystic conditions, and tissue hypertrophy.

The term “a disease or condition of the immune/hematopoietic system” asused herein refers to a disease or condition selected from the groupconsisting of: anemia, pancytopenia, leukopenia, thrombocytopenia,leukemia, Hodgkin's disease, non-Hodgkin's lymphoma, acute lymphocyticanemia (ALL), plasmacytomas, multiple myeloma, Burkitt's lymphoma,arthritis, asthma, AIDS, autoimmune disease, rheumatoid arthritis,granulomatous disease, immune deficiency, inflammatory bowel disease,sepsis, neutropenia, neutrophilia, psoriasis, immune reactions totransplanted organs and tissues, systemic lupus erythematosus,hemophilia, hypercoagulation, diabetes mellitus, endocarditis,meningitis, Lyme Disease, Celiac disease (gluten sensitivity) andallergies.

The term “a disease or condition of the reproductive system” as usedherein refers to a disease or condition selected from the groupconsisting of: cryptorchism, prostatitis, inguinal hernia, varicocele,leydig cell tumors, verrucous carcinoma, prostatitis, malacoplakia,Peyronie's disease, penile carcinoma, squamous cell hyperplasia,dysmenorrhea, ovarian adenocareinoma, Turner's syndrome, mucopurulentcervicitis, Sertoli-Leydig tumors, ovarian cancer, uterine cancer,pelvic inflammatory disease, testicular cancer, prostate cancer,Klinefelter's syndrome, Young's syndrome, premature ejaculation,diabetes mellitus, cystic fibrosis, Kartagener's syndrome, testicularatrophy, testicular feminization, anorchia, ectopic testis,epididymitis, orchitis, gonorrhea, syphilis, testicular torsion, vasitisnodosa, germ cell tumors, stromal tumors, dysmenorrhea, retroverteduterus, endometriosis, fibroids, adenomyosis, anovulatory bleeding,amenorrhea, Cushing's syndrome, hydatidiform moles, Asherman's syndrome,premature menopause, precocious puberty, uterine polyps, dysfunctionaluterine bleeding, cervicitis, chronic cervicitis, mucopurulentcervicitis, cervical dysplasia, cervical polyps, Nabothian cysts,cervical erosion, cervical incompetence, cervical neoplasms,pseudohermaphroditism, and premenstrual syndrome.

The term “a disease or condition of the musculoskeletal system” as usedherein refers to a disease or condition selected from the groupconsisting of bone cancers (e.g., osteochondromas, benign chondromas,chondroblastoma, chondromyxoid fibromas, osteoid osteomas, giant celltumors, multiple myeloma, osteosarcomas), Paget's Disease, rheumatoidarthritis, systemic lupus erythematosus, osteomyelitis, Lyme Disease,gout, bursitis, tendonitis, osteoporosis, osteoarthritis, musculardystrophy, mitochondrial myopathy, cachexia, and multiple sclerosis.

The term “a disease or condition of the cardiovascular system” as usedherein refers to a disease or condition selected from the groupconsisting of: myxomas, fibromas, rhabdomyomas, cardiovascularabnormalities (e.g., congenital heart defects, cerebral arteriovenousmalformatiens, septal defects), heart disease (e.g., heart failure,congestive heart disease, arrhythmia, tachycardia, fibrillation,pericardial Disease, endocarditis), cardiac arrest, heart valve disease(e.g., stenosis, regurgitation, prolapse), vascular disease (e.g.,hypertension, coronary artery disease, angina, aneurism,arteriosclerosis, peripheral vascular disease), hyponatremia,hypernatremia, hypokalemia, and hyperkalemia.

The term “a disease or condition described as mixed fetal” as usedherein refers to a disease or condition selected from the groupconsisting of: spina bifida, hydranencephaly, neurofibromatosis, fetalalcohol syndrome, diabetes mellitus, PKU, Down's syndrome, Patausyndrome, Edwards syndrome, Turner syndrome, Apert syndrome, Carpentersyndrome, Conradi syndrome, Crouzon syndrome, cutis laxa, Cornelia deLange syndrome, Ellis-van Creveld syndrome, Holt-Oram syndrome,Kartagener syndrome, Meckel-Gruber syndrome, Noonan syndrome,Pallister-Hall syndrome, Rubinstein-Taybi syndrome, Scimitar syndrome,Smith-Lemli-Opitz syndrome, thrombocytopenia-absent radius (TAR)syndrome, Treacher Collins syndrome, Williams syndrome, Hirschsprung'sdisease, Meckel's diverticulum, polycystic kidney disease, Turner'ssyndrome, and gonadal dysgenesis, Klippel-Feil syndrome, Ostogenesisimperfecta, muscular dystrophy, Tay-Sachs disease, Wilm's tumour,neuroblastoma, and retinoblastoma,

The term “a disease or condition of the excretory system” as used hereinrefers to a disease or condition selected from the group consisting of:bladder cancer, prostate cancer, benign prostatic hyperplasia, bladderdisorders (e.g., urinary incontinence, urinary retention, urinaryobstruction, urinary tract infections, interstitial cystitis,prostatitis, neurogenic bladder, hematuria), renal disorders (e.g.,hydronephrosis, proteinuria, renal failure, pyelonephritis,urolithiasis, reflux nephropathy, and unilateral obstructive uropathy).

The term “a disease or condition of the neural/sensory system” as usedherein refers to a disease or condition selected from the groupconsisting of: brain cancer (e.g., brain stem glioma, brain tumors,central nervous system (Primary) lymphoma, central nervous systemlvmphoma. cerebellar astrocyroma, and cerebral astrocytoma,neurodegenerative disorders (e.g., Alzheimer's Disease.Creutzfeldt-Jakob Disease, Parkinson's Disease, and Idiopathic PresenileDementia), encephalomyelitis, cerebral malaria, meningitis, metabolicbrain diseases (e.g., phenylketonuria and pyruvate carboxylasedeficiency), cerebellar ataxia, ataxia telangiectasia, and AIDS DementiaComplex, schizophrenia, attention deficit disorder, hyperactiveattention deficit disorder, autism, and obsessive compulsive disorders.

The term “a disease or condition of the respiratory system” as usedherein refers to a disease or disorder selected from the groupconsisting of: cancers of the respiratory system such as larynx cancer,pharynx cancer, trachea cancer, epiglottis cancer, lung cancer, squamouscell carcinomas, small cell (oat cell) carcinomas, large cellcarcinomas, adenocarcinomas, allergic reactions, cystic fibrosis,sarcoidosis, histiocytosis X, infiltrative lung diseases (e.g.,pulmonary fibrosis and lymphoid interstitial pneumonia), obstructiveairway diseases (e.g., asthma, emphysema, chronic or acute bronchitis),occupational lung diseases (e.g., silicosis and asbestosis), pneumoniaand pleurisy.

The term “a disease or condition of the endocrine system” as used hereinrefers to a disease or condition selected from the group consisting of:cancers of endocrine tissues and organs (e.g., cancers of thehypothalamus, pituitary gland. thyroid gland, parathyroid glands,pancreas, adrenal glands, ovaries, and testes), diabetes (e.g., diabetesinsipidus, type I and type II diabetes mellitus), obesity, disordersrelated to pituitary glands (e.g., hyperpituitarism, hypopituitarism,and pituitary dwarfism), hypothyroidism. hyperthyroidism, goiter,reproductive disorders (e.g., male and female infertility), disordersrelated to adrenal glands (e.g., Addison's Disease, corticosteroiddeficiency, and Cushing's Syndrome), kidney cancer (e.g., hypermephroma,transitional cell cancer, and Wilm's tumour), diabetic nephropathy,interstitial nephritis, polycystic kidney disease, glomerulonephritis(e.g., 1 gM mesangial proliferative glomerulonephritis andglomerulonephritis caused by autoimmune disorders; such as Goodpasture'ssyndrome), and nephrocalcinosis.

The term “a disease or condition of the digestive system” as used hereinrefers to a disease or condition selected from the group consisting of:ulcerative colitis, appendicitis, Crohn's disease, hepatitis, hepaticencephalopatby, portal hypertension, cholelithiasis, cancer of thedigestive system (e.g., biliary tract cancer, stomach cancer, coloncancer, gastric cancer, pancreatic cancer, cancer of the bile duct,tumors of the colon (e.g., polyps or cancers), and cirrhosis),pancreatitis, ulcerative disease, pyloric stenosis, gastroenteritis,gastritis, gastric atrophy, benign tumors of the duodenum, distension,irritable bowel syndrome, malabsorption, congenital disorders of thesmall intestine, bacterial and parasitic infection, megacolon,Hirschsprung's disease, aganglionic megacolon, acquired megacolon,colitis, anorectal disorders (e.g., anal fistulas, hemorrhoids),congenital disorders of the liver (e.g., Wilson's disease,hemochromatosis, cystic fibrosis, biliary atresia, and alpha1-antitrypsin deficiency), portal hypertension, cholelithiasis, andjaundice.

The term “a disease or condition of the connective/epithelial” as usedherein refers to a disease or condition selected from the groupconsisting of: connective tissue metaplasia, mixed connective tissuedisease, focal epithelial hyperplasia, epithelial metaplasia,mucoepithelial dysplasia, graft v. host disease, polymyositis, cystichyperplasia, cerebral dysplasia, tissue hypertrophy, Alzheimer'sdisease, lymphoproliferative disorder, Waldenstron's macroglobulinemia,Crohn's disease, pernicious anemia, idiopathic Addison's disease,glomerulonephritis, bullous pemphigoid, Sjogren's syndrome, diabetesmellitus, cystic fibrosis, osteoblastoma, osteoclastoma, osteosarcoma,chondrosarcoma, osteoporosis, osteocarthritis, periodontal disease,wound healing, relapsing polychondritis, vasculitis, polyarteritisnodosa, Wegener's granulomatosis, cellulitis, rheumatoid arthritis,psoriatic arthritis, discoid lupus erythematosus, systemic lupuserythematosus, scleroderma. CREST syndrome, polymyositis,dermatomyositis, mixed connective tissue disease, relapsingpolychondritis, vasculitis, Henoch-Schonlein syndrome, erythema nodosum,polyarteritis nodosa, temporal (giant cell) arteritis, Takayasu'sarteritis, Wegener's granulomatosis, Reiter's syndrome, Behcet'ssyndrome, ankylosing spondylitis, cellulitis, keloids, Ehler Danlossyndrome, Marfan syndrome, pseudoxanthoma elasticum, osteogenesisimperfecta, chondrodysplasias, epidermolysis bullosa. Alport syndromeand cutis laxa.

The phrase “ghrelin-associated diseases and disorders” refers to anycondition that can be treated prevented or ameliorated through themodulation of ghrelin activity. These include conditions that areenhanced, exacerbated or stimulated by ghrelin, for example, growthhormone release or drive to eat. The physiological actions of ghrelinare considered to include, by way of example, the stimulation of growthhormone release, the stimulation of hormone secretion from lactotrophsand corticotropes, orexigenic and cardiovascular actions,anti-proliferative effects on thyroid and breast tumors and regulationof gastric motility and acid secretion through vagal mediation. (See WO2005021026).

Where the definition of terms departs from the commonly used meaning ofthe term, applicant intends to utilize the definitions provided herein,unless specifically indicated.

The invention will now be further described by reference only to thefollowing non-limiting examples. It should be understood, however, thatthe examples following are illustrative, and should not be taken in anyway as a restriction on the generality of the invention describedherein. In particular, while the invention is described in detail inrelation to the use of a specific H. pylori strain, it will be clearlyunderstood that the findings herein are not limited to this strain.

EXAMPLE 1 Vectors and Transgenic H. pylori Organisms for StableExpression of Foreign Proteins

The genetic manipulation of H. pylori is uncommon. The present exampledemonstrates the utility of the invention for providing a geneticallytransformed Helicobacter, particularly transformed H. pylori. Thetransformed bacterium are prepared using plasmids and plasmid vectorsderived from Helicobacter, which have had been subject to priormanipulation in a non-Helicobacter organism, such as E. coli.

Several H. pylori plasmids described in the literature can besuccessfully converted to H. pylori/E. coli shuttle vectors. Manystrains of E. coli have been reported to be naturally competent for DNAuptake. Resistance markers for streptomycin, rifampin and metronidazolehave also been successfully transformed into most strains of H. pylori.However, while plasmid DNA from E. coli and other organisms can beintroduced into H. pylori, these plasmids cannot be stably maintained.Moreover, H. pylori plasmids cannot be transformed into E. coli orHelicobacter species. Accordingly, H. pylori shuttle vectors must beconstructed.

Two plasmids from H. pylori are illustrated in the schematics shown inFIGS. 1 and 2. Vectors pHPAI (2.8 kb) (FIG. 1) and pHP3 (3.4 kb) (FIG.2) have been sequenced, and it has been revealed that pHPAI replicatedvia the theta mode of plasmid replication. In contrast to rolling-circlereplicating plasmids, theta plasmids do not generate single-stranded DNAintermediates during replication and are thus more stable vectorcandidates because they are less prone to illegitimate recombination.Furthermore, the pHPAI origin of replication (ori) contains a series ofdirect repeat sequences (termed “iterons”) that are involved inreplication control and maintaining stable copy number. Vector pHP3shares many of these features. The nucleotide sequences for these twovectors are shown below.

Plasmid pHP1 shown in double stranded form (top strand is (+strand) SEQID: 1; bottom strand is (−strand) (SEQ ID NO: 2)GTCATGCGCGTTGTTTTTAATTACATTTTAAACAACTTGTTGTTGTTTTT ACATGTTTTACTCGC      65 CAGTACGCGCAACAAAAATTAATGTAAAATTTGTTGAACAACAACAAAAATGTACAAAATGAGCG ATGCGCGCGCGTGAGGGATTGGGGGTTGCAACCCCCTAAATAACGAAGCTGTAGGGTTTCTCATT       130TACGCGCGCGCACTCCCTAACCCCCAACGTTGGGGGATTTATTGCTTCGA CATCCCAAAGAGTAATTTGTGGTGAAAATGAATAAAACAGAACTTCTTGCCAACACTAACAGAAC TTCTTGCCAACACTA      195 AAACACCACTTTTACTTATTTTGTCTTGAAGAACGGTTGTGATTGTCTTGAAGAACGGTTGTGAT ACAGAACTTCTTGCCAACACTAACAGAACTTCTTGCCAACACTAACAGAACTTCTTTATTTTAAA       260TGTCTTGAAGAACGGTTGTGATTGTCTTGAAGAACGGTTGTGATTGTCTT GAAGAAATAAAATTTGTTATGATTATTAACAATTTTTAGACATAATAACAGCGTGTGAAGATACT TTTGTAGCGGTATTT      325 CAATACTAATAATTGTTAAAAATCTGTATTATTGTCGCACACTTCTATGAAAACATCGCCATAAA CCTATGTGCGGCAAAATTTGGAGCAATTAGCTTGACTTGGTTGAGTTAGTGGGTTGGAGGATAGA       390GGATACACGCCGTTTTAAACCTCGTTAATCGAACTGAACCAACTCAATCA CCCAACCTCCTATCTGAGGGCGACACCTCGTTAGGAGGTATCAATGTGAAAGTATTTGTCGTATT AGTTCTAGTATTAGT      455 CTCCCGCTGTGGAGCAATCCTCCATAGTTACACTTTCATAAACAGCATAATCAAGATCATAATCA AATTCTCGCACAATTGCTATATTAGGCTTATTCGTGGTCTAACCCCTTGTTTATGGGGGTTGGCT       520TTAAGAGCGTGTTAACGATATAATCCGAATAAGCACCAGATTGGGGAACA AATACCCCCAACCGACGTTATAAGCATACTGATACGATCACACTTATTATACACCAAAAGATAAG GAGTATAGAGTGGAA      585 GCAATATTCGTATGACTATGCTAGTGTGAATAATATGTGGTTTTCTATTCCTCATATCTCACCTT TTTGATCAATCAGATTTACAAAAAGCGTTGAAAATATTAGATACACTCCCACAAACCCCACAAAT       650AAACTAGTTAGTCTAAATGTTTTTCGCAACTTTTATAATCTATGTGAGGG TGTTTGGGGTGTTTATGAGCTACAAAAACAAGAAATACAAAACCGCATCAACAAAATAACAGAGA CAATCATTAAAGAAT      715 ACTCGATGTTTTTGTTCTTTATGTTTTGGCGTAGTTGTTTTATTGTCTCTGTTAGTAATTTCTTA TACTATCAAAGCATGAAATCAAGAAAGAAGAACTAGAACCCACTCTAACCCCAAAACCCACACCA       780ATGATAGTTTCGTACTTTAGTTCTTTCTTCTTGATCTTGGGTGAGATTGG GGTTTTGGGTGTGGTCTCAAAGAGCCACAAACCACCCCAACACCATGCAAAGATTTAGTGGTTAG CACCCCTAAAGATAA      845 GAGTTTCTCGGTGTTTGGTGGGGTTGTGGTACGTTTCTAAATCACCAATCGTGGGGATTTCTATT AACCTAATATCACCTACCACAATAACGCTAATAAGGTCAATCTAGGGAAATTGAGCGAAAGGGAA       910TTGGATTATAGTGGATGGTGTTATTGCGATTATTCCAGTTAGATCCCTTT AACTCGCTTTCCCTTGCCAATCTTTTATTCGCTATTTTTCAAAAACTCAAAGCCCAAGGGAATAC CCTCATTCGTTTTGA      975 CGGTTAGAAAATAAGCGATAAAAAGTTTTTGAGTTTCGGGTTCCCTTATGGGAGTAAGCAAAACT ACCGCAAGATTTGAAACGCATGCTAAACATAGATATTTCTAATGAGCGCTTATCAGAAGTCGTTA       1040TGGCGTTCTAAACTTTGCGTACGATTTGTATCTATAAAGATTACTCGCGA ATAGTCTTCAGCAATTTAAGCTGTGGGATAGCATTAAAACCGCTGATTTTTGGAAAATTAGCGAA ACCGAAACTTCAATC      1105 AATTCGACACCCTATCGTAATTTTGGCGACTAAAAACCTTTTAATCGCTTTGGCTTTGAAGTTAG ATTCAAGAAAATTACATGCTTTTTAGTCGGTGTAAAATTGAATTGAACAAACCGAGTAAAGATTT       1170TAAGTTCTTTTAATGTACGAAAAATCAGCCACATTTTAACTTAACTTGTT TGGCTCATTTCTAAAGAAGTATTTAGAAATCCAACTCAACGATAACTATCAAGACTTACTCAACA ATCTGGGCATGGGTC      1235 CTTCATAAATCTTTAGGTTGAGTTGCTATTGATAGTTCTGAATGAGTTGTTAGACCCGTACCCAG AATACACTTCTTTCAATCTGTTAGAATTTCAAAGAGTGAGGGGTAAATACGCTAAAACGCTCTAT       1300TTATGTGAAGAAAGTTAGACAATCTTAAAGTTTCTCACTCCCCATTTATG CGATTTTGCGAGATACGCTTGCTCAAGCAATACAAAAGCACAGGGATTTTGAGCGTGGAATGGAC TCAATTCAGGGAGCT      1365 GCGAACGAGTTCGTTATGTTTTCGTGTCCCTAAAACTCGCACCTTACCTGAGTTAAGTCCCTCGA TTTAGACATTCCAAAAGACTACAAAATGGAAAACATCGATCAAAAAGTCTTAACCCCCTCTCTCA       1430AAATCTGTAAGGTTTTCTGATGTTTTACCTTTTGTAGCTAGTTTTTCAGA ATTGGGGGAGAGAGTAAGAACTCAGAAAAATCTACCCTTTTGAACACTTGAGCTATAAAAAAGAA CGCAAAAGCCATTAC      1495 TTCTTGAGTCTTTTTAGATGGGAAAACTTGTGAACTCGATATTTTTTCTTGCGTTTTCGGTAATG AAGCGCAAAGTAACCCACATTGATTTTTATTTTGAGCAATTTCCTTAAGGCGAAAATAAGAAACA       1560TTCGCGTTTCATTGGGTGTAACTAAAAATAAAACTCGTTAAAGGAATTCC GCTTTTATTCTTTGTAAACAAAGCCGACAAGCAACGCGCTCAAAGGGACATCAAGCTTGTAGCAT GGGATATTCACAACC      1625 TTTGTTTCGGCTGTTCGTTGCGCGAGTTTCCCTGTAGTTCGAACACGTACCCTATAAGTGTTGG AAATCGCTAAAAGAAACGCAAAAGCCACTATGGAAGCTAGGTTTCTTGAATTGAAAACTTTGATC       1690TTTAGCGATTTTCTTTGCGTTTTCGGTGATACCTTCGATCCAAAGAACTT AACTTTTGAAACTAGGGCTATCAGTTCAGGAACAATGACAGTAGGAACAAATTAAAGATTGACAA CACCACTTTTGAAAG      1755 CCGATAGTCAAGTCCTTGTTACTGTCATCCTTGTTTAATTTCTAACTGTTGTGGTGAAAACTTTC AATCAAATGTATTTACATGTATCTTAACCCTAAAAATAAGCATAACCCCCAAAAATTCCTTGTAT       1820TTAGTTTACATAAATGTACATAGAATTGGGATTTTTATTCGTATTGGGGG TTTTTAAGGAACATACCAACAAGACATTCGCATTGGAACTACTATATATCAATAGATACAGCCTA AAAAAAAGACAACTT      1885 GGTTGTTCTGTAAGCGTAACCTTGATGATATATAGTTATCTATGTCGGATTTTTTTTCTGTTGAA GCTAGAAGAATTTAACCCCCCAAAATCCACCCTATCACCAACGAACCTATCAAGGAATTTGCAGA       1950CGATCTTCTTAAATTGGGGGGTTTTAGGTGGGATAGTGGTTGCTTGGATA GTTCCTTAAACGTCTATACATCGGCAAAACGATTAACATCACCAACTTCAATGTGGATCAATGCC ATGAGGGAATCAGCA      2015 TATGTAGCCGTTTTGCTAATTGTAGTGGTTGAAGTTACACCTAGTTACGGTACTCCCTTAGTCGT ACTACCTGACAATCACTAGGATCGTGAACTGGACGTAATCGGATCTGTATTTGGTCCAGATGTGG       2080TGATGGACTGTTAGTGATCCTAGCACTTGACCTGCATTAGCCTAGACATA AACCAGGTCTACACCATAAGCCTGGGACTTCTCAAGCCTTTCATTGCTAAAGTGAGAAAATTTGG GGATTGGTTCAAGAA      2145 TATTCGGACCCTGAAGAGTTCGGAAAGTAACGATTTCACTCTTTTAAACCCCTAACCAAGTTCTT CACTACAGGTGAAAAGACAGATGCATGCTGACTAAACTCATAGAAAAACTGAATCACGAAAGAAA       2210GTGATGTCCACTTTTCTGTCTACGTACGACTGATTTGAGTATCTTTTTGA CTTAGTGCTTTCTTTGAATGCAAGCAGAAAACAAACACCTAAAAGAACAAGGACTAGAAAAAATC TACACTCAAAAAGAC      2275 CTTACGTTCGTCTTTTGTTTGTGGATTTTCTTGTTCCTGATCTTTTTTAGATGTGAGTTTTTCTG TACGAGCAGTTAAAAGAACAGCATTTGAAAGAAATTGAAGCACTCAAAAAAGAAATCCAAAAAAC       2340ATGCTCGTCAATTTTCTTGTCGTAAACTTTCTTTAACTTCGTGAGTTTTT TCTTTAGGTTTTTTGCAAGCAAGAAACATACACGCAACCAAAAGAATGTAGCCATTTAGCGCATT CTTTTAGCCCTAATT      2405 GTTCGTTCTTTGTATGTGCGTTGGTTTTCTTACATCGGTAAATCGCGTAAGAAAATCGGGATTAA CATTCTTTCAATCAAAATCCGACTAATTCATCGGCTAAACGCTAAAAATCGCTTAAAACGAAAAA       2470GTAAGAAAGTTAGTTTTAGGCTGATTAAGTAGCCGATTTGCGATTTTTAG CGAATTTTGCTTTTTTACAAAGCAAAAAACTTCATTCCCCTTTTAGTCGTTAACCATTTAGCCAA TCTAACTAGTTTAGC      2535 ATGTTTCGTTTTTTGAAGTAAGGGGAAAATCAGCAATTGGTAAATCGGTTAGATTGATCAAATCG ATCTAAAGGCGAATCTATCTTGTGTTAGACATCCAACCTTACCAAAACCGCAGAGCGAGCTTAAG       2600TAGATTTCCGCTTAGATAGAACACAATCTGTAGGTTGGAATGGTTTTGGC GTCTCGCTCGAATTCAGAGATTCAAGCGGTTTTGCACGATTGTTTGCTGCCAAGAAAACCAACAA GCGAAGTAAGGCGCA      2665 TCTCTAAGTTCGCCAAAACGTGCTAACAAACGACGGTTCTTTTGGTTGTTCGCTTCATTCCGCGT TAGACAAAAGCGCATCGCAGTTTGAAAGCGTAGGCGTCAGAAGTGGTTTGCGTTAGAATCAAACA       2730ATCTGTTTTCGCGTAGCGTCAAACTTTCGCATCCGCAGTCTTCACCAAAC GCAATCTTAGTTTGTAGATAGCGCAAACCTGGCGTTAGGCTAAAAAACCCCTAAAAACTAAAACC CCAAAATATGTAGTGC      2796 TCTATCGCGTTTGGACCGCAATCCGATTTTTTGGGGATTTTTGATTTTGGGGTTTTATACATCACG

Plasmid pHP3 shown in single stranded form (SEQ ID NO: 3):TCTACACAATTAACAATCTTTAGCTACAATAACAGCGTGTGAAGATGCTT TCACAGCGGT       60ATTTCCTATGTGCGGCAAAATTTGGAGCAATTAACTTGACTTGGTTGGGT TAGTGGGTTG       120GAGGATAGAGAGGGCGACACCTCGTTAGGAGGTATCAATGTGAAAGTATT TGTCGTATTA       180GTTCTAGTATTAGTAATTCTCGCACAATTGCTATATTAGGCTTATTTGTG GTCTAACCCC       240TTGTTTATGGGGGTTAGATCCTTATAAGCATACTGATACGATCACACTTA TTATACACCA       300AAAGATAAGGAGTATAGAGTGGAATTTGATCAATTAGAATCACAAAGATC AGACTTACAA       360AAAGTGTTAAAAGAATTAGATACACTCCCAAAAACCCCACAAATTGAGCT ACAAAAACAA       420GAAATACAAAACCGCATCAACAAAATAACAGACACAATCATTAAAGAATT ACTATCAAAA       480CATGAAATCAAAAAAGAAGAACTAGAACCCACTCTAACCCCAAAACCCAC ACCAACAAAA       540GAGCCACAAACCACCCCCACACCATGCAAAAATTTAGTGGTTAGCACCCC TAAAGATAAA       600ACCTATATCACCTACCACAATAACGCTAATAAGGTCAATCTAGGGAAATT GAGCGAAAGG       660GAAGCCAATCTTTTATTCGCTATTTTTCAAAGGCTTAAAGATCAAGGGAA TACCCTCATT       720CGTTTTGAACCGCAAGATTTAAAACGCATGATCATGGTCAAATCCAACTT AACCAACAGG       780CAATTATTGCAAGTCTTAAAAAATTTGCTTGACAACATTAGCGGTGCTAA TTTTTGGATC       840AATTAGAGAGCATGTTGAAAATGGCGAAATCTATGAAGATCACACTAGCT ACATGCTTTT       900CAAACAATTTGAAATCCGCATCCATAAGCCAACACAAACTATAGAATACT TAGATGTCCA       960ACTCAATGATAGCTATCAATACTTGCTCAACAATCTAGGAATGGGCGGTC AATACACTTC       1020TTTCAATCTCTTAGAATTTCAAAGGGTGAGGGGCAAATAGTGAGAGCGTT AAATTTCCCC       1080CCCCTATTCCCCTTAAAAAGGACCCTTATCCCAGGGAATTTTTGGCCCCA ATACAATTAG       1140GGCCAAAAACCCGGTCCCTTCCATGGCTTAACCAACCCAATTGGGGGATT CCAATTTCCC       1200CTGGATGGGAATAACCCAAGGCTTTTTTTGAAAATTCCACCTACCATTTG GTCCAAAATT       1260GGATGGACAATTCCAAATTCCAAATCTTCTTTTCCAAGAATGGGGGCCAA CCCTTGACAA       1320ACTCCTTAAACCTTTTCATTCGGCTAAAAGGTTGAAAAACATTTGGAAGA TTTGGTTTAA       1380GGAAATATTTATCGGGTGAAAAGACCAGATGCATGGCTAACTTAAACTCC ATAGAAAAAC       1440TGAATCACGAAAGAAAGAATGCTATCAAAAATGGCATTTACCACTTGATC CAAATCAAAT       1500TTTCTTACAACTCCAATCGCATTGAAGGAAGCGGTTTGACTTATGAACAA ACCGCTCATA       1560TTTTTGACAAATCCGTTCTCATAACTGAAAAAAACACCAATATCAAACTT GATGATATTT       1620TTGAAACTATCAATCATTTTGAATGCGTGAATTACTTGCTTGAAAGCTAT AAAGAACCTT       1680TGAGTTTAGAATACTTTAAGAATTTACACAAAATCTTGAAAAAGAATTGT TCTGATGAAG       1740TTATTGGTGATTTTAAAAAACGCCCTAATTTTGTAGGCAATAGCGCCACA ACAAGACCCA       1800AATTAGTTGAAAGCGAATTGACAAATCTTGTGAAAAATTATCAACGCAAC CTTGAAGTGA       1860GTTTGAAAAACAATATCATGCCTTTCATCATAGAAAACGAACACAAAGCC TTTTACTACA       1920GGGGCATCAAAGAATATGACAACACAAAAGGCTACTTGAAAGACACCATT TTGCAAAGTC       1980AAGACAATTTCAATGAAATGGTTAGCTATTTCTTTTCTTGAGTGAAACCG CTTATTTTTG       2040CTTGTGTGCTTTTGTTTTTTGCGTTTTTAGTTGTAGGTGGTAAGAAATAT CGGTTTTTTG       2100CTTTTCGTTGGTTGTAGGCGATTTTAGATAGCAAAAAACAGCTAAAAAAT CCAAGCAACC       2160TAATTGATTTCAAACCAACTTCATTTCCCTTTTAGTCGTTAGCCATTTAG CCAATCTAAC       2220TAGTTTAGCATCTAAAAGCGCATATAACTTGAGTTAGCAATCCAACCAAT ACTAAAACCG       2280CCTAGCGAAGCGTTAGCGAGCAAAATAAGCGGTTTTAGACCGATTGTTTG CTGACAAGCA       2340AACACCAATAAGCGAGCGTTAGCGAGCATGGACAAAAGCGCATCGCAGTT TGAAAGCGTA       2400GGCGTTAGCCGAAGCTGTTTTGCGTAAGCAAATCAAACAAGATAGCGCAA GCCGAGGTGC       2460AGCCCAAGAATTTGAATTAATCCATGCGGTGTTTAGGGCGTTTTAGCGTG ATCGCTTTAT       2520TACATGTTTTAACAGCATGCTGTTTTTTACATGTTTTACTCGCATGCGCG CGCGCTAGG       2580TATTGGTGGTTGGAATAGCCTAAATAACGCAGCTGTATGGTTTCTCATTT TTCGGTGACA       2640ATGAATAAGGGGTAGTTCTTGCGAGTCATAAGTGTAGTTCTTGCGAGTCA TAAGTGTAGT       2700TCTTGCGAGTCATAAGTGTAGTTCTTGCGAGTCATAAGTGTAGTTCTCTT CACAATATCT       2760ACACAATTCACAATCTCTAGCTACAATAACAGCGTGTGAAGATGCTTTCA CAGCGGTATT       2820TCCTATGTGCGGCAAAATTTGGAGCAATTAGCTTTAAAAGCTAGTGGGTT GGGAGTTTGT       2880AGCGGGTATGCACTCCGTTAGGAGGCACACCATGAAAGCATTTTTGATAG TAGTGATTTT       2940AGTGGTAATCTTGACACAGCCACTATATTAAAACCTTAGCGTTTTAATAA CCCTTATAAG       3000TCCGCCAAGACTTCTTAAGGTTTCACTCCTGTTATTATATCGTCTTTTGA AAAATAAGC       3060ATTAAAAGGCGCTTAAATGCCCATGAATACGAATTTTGAACAGCTTAGAA AACAAGAATT       3120GGAATTACGAAAATTATTAGAAGAATTAGAAACGCTCCCACAAACCCCAC AAATTAAACT       3180GCAAAAACAAAAAATACAAACTTACATAGACAAGATAACACCAAGTATTT TGAGCGGTTT       3240TGATCAAAAATTCAAAGAAATTATAGAAAATCTATCAAATGAATTTGAAA AAGAAAAATC       3300CACACCACTCAAAGAGCCACAAACCACCCCCACACCATGCAAAGATTTAG TGGTTAGCAC       3360CCCTAAAGATAACACCTATACCACCTACCACAATAACGCTAATAAGGTCA ATCTAGGGAA       3420ATTGAGCGAAAGGGAAGCCAATCT       34440

An additional nucleotide sequence that was cloned is provided at SEQ IDNO: 4, which includes a 135 bp segment that encodes a peptide of 45amino acids (SEQ ID NO: 5). This smaller 45 amino acid peptide is animmunogenic polypeptide of the Hepatitis C virus (HCV) core antigen. Thenucleic acid sequence encoding the 45 amino acid peptide is shown belowwith the indicated 135 nucleotides underscored (SEQ ID NO: 5).CATGAGCACG AATCCTAAAC CTCAAAGAAA SEQ ID NO:4 AACCAAACGT AACACCAACCGTCGCCCACA GGACGTCAAG TTCCCGGGTG GCGGTCAGAT CGTTGGTGGA CTTTACTTGTTGCCGCGCAG GGGCCCTAGA TTGGGTGTGC GCGCGACGAG GAAGACTTCC GAGCGGTCGCAACCTCGAGG TAGACGTCAG CCTATCCCCA AGGCACGTCG GCCCGAGGGC AGGACCTGGGCTCAGCCCGG GTACCCTTGG CCCCTCTATG GCAATGAGGG TTGCGGGTGG GCGGGATGGCTCCTGTCTCC CCGTGGCTCT CGGCCTAGCT GGGGCCCCAC AGACCCCCGG CGTAGGTCGCGCAATTTGGG TAAGGTCATC GATACCCTTA CGTGCGGCTT CGCCGACCTC ATGGGGTACATACCGCTCGT CGGCGCCCCT CTTGGAGGCG CTGCCAGGGC CCTGGCGCAT GGCGTCCGGGTTCTGGAAGA CGGCGTGAAC TATGCAACAG GGAACCTTCC TGGTTGCTCT TTCTCTATCTTCCTTCTGGC CCTGCTCTCT TGCCTGACTG TGCCCGCTTC AGCCTACCAA AATCCTAAACCTCAAAGAAA AACCAAACGT SEQ ID NO:5 AACACCAACC GTCGCCCACA GGACGTCAAGTTCCCGGGTG GCGGTCAGAT CGTTGGTGGA GTTTACTTGT TGCCGCGCAG GGGCCCTAGATTGGGTGTGC GCGCG

The nucleic acid of SEQ ID NO: 4 was cloned into the hopE gene (SEQ IDNO: 6, shown below), of H. pylori 26695 at nt504 of SEQ ID NO: 4 (notedin bold/underscore; corresponding to amino acid residue 168 of theprotein product) so that the expressed product would be located as partof the surface exposed loop of the HopE gene product. This construct,designated as vector pTMI03-8 (FIG. 3) was expressed on the surface ofE. coli. ATGCCATAGC ATTTTTATCC ATAAGATTAG CGGATCCTAC CTGACGCTTTTTATCGCAAC TCTCTACTGT TTCTCCATAC CCGTTTTTTG GGCTAACAGG AGGAATTAAC C 1ATGGAATTTA TGAAAAAGTT TGTAGCTTTA GGGCTTCTAT CCGCAGTTTT 51 AAGCTCTTCGTTGTTAGCCG AAGGTGATGG TGTTTATATA GGGACTAATT 101 ATCAGCTTGG ACAAGCCCGTTTGAATAGTA ATAAAATAA TACAGGGGAT 151 TGCACAGGGA GTGTTGTAGG TTGCCCCCCAGGTCTTACCG CTAATAAGCA 201 TAATCCAGGA GGCACCAATA TCAATTGGCA TGCTAAATACGCTAATGGGG 251 CTTTGAATGG TCTTGGGTTG AATGTGGGTT ATAAGAAGTT CTTCCAGTTC301 AAGTCTTTTG ATATGACAAG CAAGTGGTTT GGTTTTAGAG TGTATGGGCT 351TTTTGATTAT GGGCATGCCA CTTTAGGCAA GCAAGTTTAT GCACCTAATA 401 AAATCCAGTTGGATATGGTC TCTTGGGGTG TGGGGAGCGA TTTGTTAGCT 451 GATATTATTG ATAACGATAACGCTTCTTTT GGTATTTTTG GTGGGGTCGC 501 TATCGGCGGT AACACTTGGA AAAGCTCAGCGGCAAACTAT TGGAAAGAGC 551 AAATCATTGA AGCTAAGGGT CCTGATGTTT GTACCCCTACTTATTGTAAC 601 CCTAACGCTC CTTATAGCAC CAAAACTTCA ACCGTCGCTT TTCAGGTATG651 GTTGAATTTT GGGGTGAGAG CCAATATTTA CAAGCATAAT GGCGTAGAGT 701TTGGCGTGAG AGTGCCGCTA CTCATCAACA AGTTTTTGAG TGCGGGTCCT 751 AACGCTACTAATCTTTATTA CCATTTGAAA CGGGATTATT CGCTTTATTT 801 AGGGTATAAC TACACTTTTTCTCGAGATCT GCAGCTGGTA CGATATGGGA SEQ ID NO:6 ATTCGAAGCT TTCTAGAACAAAAACTCATC TCAGAAGAGG ATCTGAATAG CGCCGTCGAC CATCATCATC ATCATTGAGTTTAACGGTCT CCAGCTTGGC TGTTTTGGCG GATGAGAGAA GATTTTCAGC CTGATACAGATTAAATC

Briefly, one method of accomplishing the isolation of hopE gene isamplification from H. pylori 22695 by using Taq DNA polymerase. Theupstream primer 5′-AAGGATCCGATAGGAATGTAAAGGAATGG-3′ (SEQ ID NO: 7)containing a BamHI site and the downstream primer5′-CCGAATTCTAAAGGCATGAACGCTTGCA-3′ (SEQ ID NO: 8) containing an EcoRIsite can be constructed by using a DNA synthesizer, such as thePerkin-Elmer Applied Biosystems, Inc. model 332 (ABI; Mississauga,Ontario, Canada). The resulting PCR fragment can be blunt-end clonedinto the EcoRV site in pBluescript II KS(+) in the same orientation asthe lac promoter.

PCR primers can then be designed to insert two unique restriction enzymesites into the hopE gene for insertion of the 135 bp immunogenic codingsequence from the Hepatitis C virus (HCV) core antigen. The PCRamplification using Taq DNA polymerase can be performed using atouchdown amplification procedure as follows. The PCR Thermocycler isprogrammed for an initial denaturation step of 96° C. for 4 min,followed by 18 cycles at an initial annealing temperature of 65° C. (for90 s), which is decreased by 0.5° C. for each successive cycle, antension step at 72° C. for 6 min., and denaturation at 96° C. for 1 min.Subsequent to completion the first 18 cycles, an additional 14amplification cycles can be performed by using 72° C. extension and 96°C. denaturation steps with a constant 55° C. annealing temperature. Theresulting amplicon is then purified by column, precipitated withethanol, and made blunt by digestion with the Klenow fragment of DNApolymerase. The PCR products can be digested with restriction enzyme toremove the template DNA, and religated into an appropriate vector suchas pTMI03.8 under the control of the arabinose inducible promoter, andtransformed into E. coli JM105.

Recombinant clones can be identified by using oligonucleotide primer5′-AGATCTAAGGACGTC-3′ (SEQ ID NO: 9) plus the reverse sequencing primerin PCR amplification reactions. Identified clones can be sequenced toverify that the inserted restriction endonuclease sites are in frame andthat no errors had been introduced into the hopE gene. The 135 bpimmunogenic coding sequence from the Hepatitis C virus (HCV) coreantigen can then be inserted using standard techniques.

Once vector pTMI03-8 had been created, it was transformed into E. coli,which was grown at 37° C., cells were then harvested and the expressionof the DCV insert was continued by Western Blot.

Briefly, a procedure for this is as follows. Cells are harvested fromabout 20 plates and resuspended in 20% sucrose with 50 mg of DNase I(Boehringer Mannheim) in 10 mM Tris-HCI (pH 8.0). The cells are thendisrupted with a French press at 15,000 lb/in². Broken cells areoverlaid on a sucrose step gradient of 1 ml of 70% and 6 ml of 70%sucrose in 10 mM Tris-HCI (pH 8.0). The outer membrane fraction iscollected and pelleted at 150,000×g, and the pellet is resuspended in100 ml of distilled water.

Alternatively, outer membranes from 500 ml of log-phase culture can besolubilized in 10 mM Tris-HCl (pH 8.0)-3% n-octyl-polyoxyethyleneincubated at 23° C. for 1 hour and centrifuged for 30 mm at 173,000×g.The pellet is resuspended in 10 mM Tris-HCI-3% n-octyl-polyoxyethylene-5mM EDTA (pH 8.0), incubated at 23° C. for 1 hour, and centrifuged for 30min at 173,000×g, and the supernatant collected. A Western immunoblotindicated the presence of HCV/hopE in the supernatant of the secondsolubilization step. The supernatant containing HCV/hopE is mixed withan equal volume of 0.125 M Tris-HCI (pH 6.8), 4% (wt/vol) sodium dodecylsulfate (SDS), and 20% (vol/vol) glycerol and subjected to SDS-12%polyacrylamide gel electrophoresis (PAGE). If required the HCV/hopE bandcan be excised from an unstained portion of the gel and eluted overnightat 4° C. into 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0), and 100 mMNaCl. The elution supernatant can then be run on an SDS-PAGE gel tocheck for purity and a Western immunoblot using standard techniquesundertaken. For example, isolated outer membranes can be loaded at aconcentration of 15 μg/lane. Electrophoresis is then carried out bySDS-PAGE on a discontinuous 12% polyacrylamide gel. Proteins are thenstained with Coomassie brilliant blue. For Western immunoblotting,unstained gels can be electroblotted onto immobilon-P membranes(Millipore, Bedford, Mass.). After blocking for 2 h at 2° C. with 3%bovine serum albumin (BSA; Boehringer Mannheim)-0.1% Tween 20 (Sigma) inphosphate-buffered saline (PBS), the membranes are then incubated with a1/10,000 dilution of anti-HCV rabbit antiserum in 1% BSA-0.05% Tween 20in PBS for 1 h at 37° C. The membranes are then washed with PBS andincubated with a 1/5,000 dilution of an alkaline phosphatase-conjugatedsecondary antibody (Bio-Rad, Richmond, Calif.) for 1 h at 37° C. Thebound antibodies are detected with 5-bromo-4-chloro-3-indolylphosphate(BCIP, Calbiochem. La Jolla, Calif.) and nitroblue tetrazolium (NBT,Sigma).

EXAMPLE 2 Expression Vectors and Selection of Antigens for StableExpression

Because HopE is a native protein of H. pylori, it is tolerated by thisorganism and can thus form a construct useful for expressing foreignantigens or other heterologous gene products in H. pylori. Other H.pylori/E. coli shuttle vectors that can be readily developed asdescribed above include, for example, vectors comprising two plasmid orisites and markers which are suitable for each host. Markers mightinclude genes for chloramphenicol/kanamycin resistance as well aspromoters that can be recognized by both the E. coli and H. pyloritranscriptional systems.

The requirements for replication E. coli can be achieved by using any ofa number of known E. coli plasmids (e.g., pBR322).

Constructed shuttle vectors can be tested for replication in both E.coli and H. pylori in vitro and compared to existing shuttle plasmidsdescribed in the literature.

The choice of antigen or other heterologous gene product to be expressedwould ideally be one that is not toxic to H. pylori and E. coli and thatis highly immunogenic (or possesses another desirable property) whendelivered to a selected site in a mammal. In the case of an expressedantigen for immunization of the mammal, such as site may be a mucosalsite.

As described in Example 1, hopE/HCV core antigen fusion protein can beexpressed at the E. coli surface. The product of pTMI03-S (FIG. 3) ispreferably targeted to the H. pylori outer membrane, so that it woulddisplay the HCV core antigen in a mucosal environment.

Tetanus toxoid (TT) has been studied extensively as an antigen inhumans, and immune responses to it are well characterized. TT elicitsgood mucosal immune responses when administered orally or intranasalwhen displayed on the surface of bacterial spores (Duc & Cutting, 2003,Expert Opinion Biol Ther, 3(8), 1263-70). The tetanus toxin C fragmentcan be fused to the hopE gene product as described above or engineeredto contain a membrane anchor and cell surface target sequence. Theadvantage of this system is the existence of well-characterized murinemodels for assessment of the effectiveness of the vaccination procedure,whereas; in the case of HCV where the known murine models typically relyon immune-deficient mice. The gB protein of cytomegalovirus (CMV) hasbeen shown to immunize mice against a lethal dose of an engineeredvaccine virus expressing the gB antigen. Accordingly, shuttle vectorssuch as those described herein, comprising gB antigen in place of an HCVantigen can be constructed using the protocol described in Example 1.

A number of promoters can be used in the shuttle vectors. Ideally thepromoter used is inducible either by a natural in vivo colonizationmechanism of Helicobacter or by induction with an innocuous foodstuff orchemical that can be consumed. For example, the promoter from the H.pylori histidine kinase HP 165 may be used. The promoter from the H.pylori histidine kinase HP 165 is reported to be induced by acidic pHand may be a virulence factor related to gastric mucosa colonization.The benefit of this promoter is that a construct can be made in vitro.The foreign antigen will only be expressed when exposed to the acidicenvironment of the gastric mucosa.

Other promoters include the arabinose inducible promoter used inpTMI03.S and FlaB sigma 54 promoters (Josenhans et al., 1998, EEMSMicrobial Lett., 16 1(2), 263-73), the T7 promoter used in constitutiveand inducible E. coli systems and the nir promoter of Salmonella whichis induced in anaerobic environments (Chatfield et al., 1992,Biotechnology, 10(8): 888-92). The ability of any of these promoters tofunction in H. pylori can be tested using the system developed byAngelini et al. (2004) (Plasmid, 51:101-107) that uses CAT and GFPreporters as readout of promoter activity in a H. pylori plasmid vector.

The target sites of expression will depend on the antigen or other geneproduct used. Initial studies focused on HopE proteins and fusionpolypeptides which target the expressed polypeptide (e.g., antigen) tothe cell surface of H. pylori.

Plasmid stability is also very important and, while the use ofantibiotic resistance genes as selective determinants for plasmidmaintenance is useful in vitro, is less practical in vivo. Analternative is a balanced-lethal system, for example, the asd gene thatis used inactivated in Salmonella. The asd gene, which exists nativelyin H. pylori, encodes aspartate-β-semialdehyde dehydrogenase (an enzymein biosynthetic pathway for diaminopimelic acid (DAP), an essentialcomponent of the cell wall peptidoglycan of gram-negative bacteria. Inthe absence of DAP, asd mutants undergo lysis. Since DAP is not presentin mammalian tissues, this balanced-lethal system imposes a requirementthat all living H. pylori carry the recombinant asd gene-containingplasmid.

In order use the asd gene system the genomic copy of the asd gene isinactivated using standard gene knockout protocols. This strain of H.pylori will then only grow with the supply of DAP or with a plasmid thatcontains the asd gene.

Other systems that can be used for similar purposes include E. colienterotoxin or cholera toxin (CT) as mucosal adjuvants. Adjuvants canalso be used to boost the mucosal immune response. Two such adjuvantsare CT and E. coli enterotoxin (LT) wherein expressed antigens are fusedto the LTB and CTB mutants that maintain their strong mucosal adjuvantproperties but have reduced toxicity.

EXAMPLE 3 Virulence, LD₅₀

As described in Examples 1 and 2, Helicobacter-based vectors such aspHP3 and pHP623 are capable of providing protection against infection ina mammal, such as a mouse or human. In the present example, a murinemodel is used to demonstrate the utility of using the Helicobacter-basedsystems to provide delivery of a pharmacologically active molecule ofinterest to a mammal, including a human. The murine model is employed todemonstrate the activity of a transgenic strain of H. pylori to elicit aserological response to an expressed surface antigen in vivo.

Mice are infected with wild-type H. pylori, while other mice areinoculated by gavage with temperature-sensitive H. pylori as describedin Example 2. Sera from both control and test animals are assayed forantibody and gastric histology are performed on sacrificed animals inaccordance with the schedule shown in Table 1. A mouse urea breath testcan also be used.

A 50% decrease in virulence (from 75% to 40%) was observed. Specificantibody titer increased 4 fold above baseline, indicating a serologicalresponse. Serum samples were taken at baseline, 12, 24 and 48 weeks. Atthese times 10, 10 and 20 animals were sacrificed and gastric histologyperformed.

EXAMPLE 4 Comparison of Virulence and Antigenicity of TemperatureSensitive H. pylori Strains

In order detect change in virulence related to expression/modificationof an outer membrane protein; mice were inoculated withtemperature-sensitive H. pylori as described in Example 3. An equal sizecontrol group of mice were infected with a wild type H. pylori strain.Noninvasive means were used to determine presence or absence of H.pylori. Mice were bled at 3 and 6 months for antibody determination. Atsacrifice, histology was performed to assay gastritis and confirmcolonization.

EXAMPLE 5 LD₅₀ Study to Evaluate Temperature-Sensitive H. pylori VaccineEfficacy for a Pneumococcal Antigen

In order to demonstrate the Helicobacter-based vaccine protection effectfrom a standard pathogen (pneumococcus), mice were inoculated withtemperature sensitive H. pylori by gavage. An equal sized control groupwas infected with the wild type H. pylori strain. Non-invasive meanswere used to determine presence or absence of H. pylori as described inExample 4. At 6 months post infection, all mice were givenintraperitoneal challenge with 10 times the LD₅₀ of live virulentpneumococci type 4 (˜20 CFU/mouse), as per the method of Aaberge et al.(1995, Microb. Pathog., 18:141-152).

Allowing for 75% lethality in the controls, the study has a power of 0.8to detect a 50% decrease in mortality (75% vs 50%).

EXAMPLE 6 Determination of H. pylori Status of Mice: Breath Test Method

In the present example, the urea breath test used in humans was adaptedfor use in mice.

Ten mice were fed a diet devoid of urease (uncooked soy). Mice were thenadministered 3.7 kBq^([14])C urea in 200 μl flavored citrate by gavageand placed in air-filled 2 L plastic Ziploc bags for 20 minutes. Micewere then removed without exchanging the air within the bag. Hyamine,0.1 mmol in ethanol, was then introduced and scintillant was added tothe hyamine solution and counted for 10 min or up to a count of 1,000dpm.

EXAMPLE 7 Human Studies

To confirm virulence and antibody response in humans, a strain of H.pylori like the “Baylor Strain” will be employed, and the followingcriteria will be adopted:

1. The infected individuals will have no symptoms, no more than mildhistological damage, and no evidence of infection with hepatitis virusesor HIV.

2. The isolate will be a single strain, cagA negative, and sensitive tometronidazole, clarithromycin, tetracycline, and amoxicillin.

3. Volunteers to receive a challenge will be healthy with normal gastrichistology, no history of peptic ulcer, no young children at home, noregular contact with young children, and no allergies to the antibioticsthat might be required to treat the infection.

Challenge will consist of 40 mg famotidine at bedtime followed byadministration of H. pylori in beef broth orally in the morning.Subjects are contacted daily for 14 days. A 13c-UBT is performed after 7and 14 days and endoscopy with quantitative culture and histology after2 weeks and 3 months. Antibiotics are used to eradicate the infection.

EXAMPLE 8 Development of External Chemical Marker for Detection of WildType and/or TSHP In Vivo

An example of a chemical marker that may be used for the detection ofwild type or TSHP in vivo is sulfasalazine (SSN), the structure of whichis shown in FIG. 4. Studies in germ free mice and conventional rats haveshown that intestinal bacteria are solely responsible for the diazo-bondreduction, resulting in the reductive catabolism of SSN and the releaseof sulfapyridine and 5-aminosalicylate. The enzyme(s) which catalysesthis reaction is referred to as diazoreductase(s) (synonymazoreductase(s)). Conventional rats given SSN excrete 5-aminosalicylateand sulfapyridine (and their respective conjugates) in urine and feces,whereas germ-free rats show no evidence of SSN degradation.

Several bacterial species have been shown to have diazoreductases(AZR's). Preliminary bioinformatic studies have indicated that H. pylorimay not contain the AZR gene. The presence of similar analogoussequences has also produced a negative result. Under these circumstancesa transgenic strain of H. pylori (TSHP) that has a viable and functionalazoreductase (azr+TSHP) can be used to assess the use of these markers.

Plasmid pTM103-02 is digested by EcoRI and HindIII, and ligated with theAzoreductase (AZR) gene from Bacillus subtilis treated with EcoRI andHindIII to generate a vector containing both HopE 168aa and AZR namedpTMI03-azr. This plasmid is transformed into E. coli to assess whetherexpression of HopE and the B. subtilis AZR occurs. pTMI02 when similarlytreated with full-length hepatitis C core antigen (HCCA) demonstratedtransport of HopE::HCCA to E. coli outer membrane employing westernblots and anti-HopE Abs.

Mice (n=30) are infected with the azr+TSHP by gavage and once AZRexpression in vivo to produce 5-aminosalicylate and sulfapyridine (andtheir respective conjugates) in urine and feces is established, humantrials can begin.

EXAMPLE 9 Use of Diagnex Blue as a Marker

The diagnostic agent “diagnex blue” consists of an ion exchange resin(Amberlite XE-96) conjugated with a dye (Azure-A). This test relies onthe fact that the resin-dye combination disassociates at pH less than2.5 after which the dye is absorbed and appears in the urine. Personswithout dye in the urine are achlorhydric. This principle is shown inFIG. 5.

The same principle can be used to test for H. pylori. For example, adye-resin combination that disassociates at pH>7.0 could detect ureaseif the resin was given with urea. This would produce a pH>7.0 in themucosal layer where H. pylori resides, thus releasing the dye.

Mice (n=30) are inoculated with a wild type H. pylori strain whilegerm-free mice (n=30) are used as controls (Pilot study). After anoptimal period allowing for the H. pylori to establish an activeinfection, the test group and the controls are introduced with apredetermined quantity of the resin-dye complex by gavage. This will befollowed by a urea solution. (Range 0.01M to 0.5M). The mice are kept inmetabolic cages and the excretion of the azure dye are monitored andquantified. Different ratios of the resin and urea concentrations aretested to verify the optimal combinations to be used.

EXAMPLE 10 Delivery Formulations

For purposes of delivery forms, the present example is provided todemonstrate the utility of the present invention as providing an aerosolspray preparation. In some of these embodiments, the aerosol spray maytake the form of pressurized packs or a nebulizer, with the use of asuitable propellant. In the case of a pressurized aerosol, the dosageunit can be determined by providing a valve to deliver a metered amount.The formulation would be prepared as a powder for administration byinhalation. Administration by inhalation can also be carried out byatomizing solutions or suspensions which contain the compositionsaccording to the invention.

The compositions according to the invention may also be formulated in aliquid for oral digestion for administration to a subject as anintravenous preparation.

All of the various preparations of the invention may be prepared byprocedures familiar to those skilled in the art, if appropriate, usingfurther suitable pharmaceutical auxiliaries. Compositions according tothe invention advantageously contain the species of Helicobacter, aloneor in combination with other desired ingredients.

Any of the above individual or combination of Helicobacter formulationsmay be included in a pharmaceutical composition comprising thepharmaceutically acceptable composition according to the presentinvention. The pharmaceutical compositions as described herein may be insolid (e.g. powder, particles, granules, sachets, tablets, capsulesetc.), semi-solid (gels, pastes etc.) or liquid (solutions, dispersions,suspensions, emulsions, mixtures etc) form and adapted foradministration via e.g. the gastrointestinal tract and gastric mucosa.The pharmaceutical compositions may thus be in powder or particulateform adapted to be dispersed in an aqueous medium before use.

A pharmaceutical composition in liquid form may be in the form of adispersion comprising the Helicobacter composition and an electrolytesolution such as, e.g. a composition that is adapted to physiologicalconditions e.g. a physiologically acceptable solution.

A pharmaceutical composition according to the invention may furthercomprise another therapeutically, prophylactically and/or diagnosticallyactive substance.

In another aspect, the invention relates to a pharmaceutical kitcomprising a first and a second container, the first containercomprising a recombinant Helicobacter composition comprising the plasmidand/or plasmid vector according to the invention and the secondcontainer comprising a dispersion medium for the Helicobactercomposition, accompanied by instructions for administering and/or dosingthe Helicobacter composition in the dispersion medium before use.

The Helicobacter composition according to the present inventioncontained in the kit may be in powder or particulate form.

A pharmaceutical kit according to the present invention may includeinstructions with recommendations for the time period during which theHelicobacter composition should be administered after dispersion in thedispersion medium.

EXAMPLE 11 Immune Modulation with Helicobacter—Vaccine Preparation

The TH1 response (T-helper cell type 1) is a cell mediated response.Over activity of this is a presumed cause of diseases such as rheumatoidarthritis (RA) and Lupus. In contrast, TH-2 is an antibody typeserological response characteristic of vaccines. The present exampledemonstrates the utility of the present invention for providing atechnique for achieving a TH-2 type response in an animal when treatedwith a Helicobacter-based vaccine treatment preparation according to thepresent invention.

Although Helicobacter pylori produces an antibody response (TH2), it hasbeen noted recently that the main response in the mucosa of the stomachmay be a TH1 cell mediated response. Therefore, it is envisioned thatthe invention may provide for both types of immune response whenprovided to the mucosa of an animal.

Use of the Helicobacter vectors and vector plasmid systems as describedherein may be used to invoke antibody response in an animal. By way ofexample, a system employing a gene expression cassette in a constructthat provided for the transformation of the bacterium, Clostridium, andthe subsequent secretion of a protein (S-layer protein) from the surfaceof the transformed Clostridium, this resulting in initiation of mucosalvaccination, is described in WO-0194599, which disclosure is herebyincorporated herein in its entirety. These constructs may also include asecretory leader sequence selected from ORF1, ORF3, ORF5-7, ORF7 orORF11.

In accordance with some embodiments of the vaccine, theHelicobacter-based vectors and vector plasmids may comprise a sequenceencoding a bacterial surface layer protein. A surface layer protein isdefined herein as any molecule of proteinaceous nature, including e.g.,protein, glycol-protein or lipoprotein occurring in the outer membraneof a bacterium and capable of being exposed on the surface of thebacterium. S-layer proteins may be continuously and spontaneouslyproduced in larger amounts than any other class of protein in the cell.

A process for preparation of a recombinant cell preparation comprising agram negative host cell, Clostridium, having the S-layer protein, isalso provided in WO-97/28263. The process may be modified and followedin accord with the procedures described herein to incorporate anS-protein as part of the Helicobacter constructs of the presentinvention.

Accordingly, in some of the vector and vector plasmid constructs, afusion protein is provided that comprises a Helicobacter sequence and anon-Helicobacter pharmacologically active molecule of interest. In orderto enhance the immunogenicity of a vaccine employing the Helicobacterconstructs of the present invention, the Helicobacter sequence of thefusion protein may comprise a sequence encoding an S-layer protein.Bacillus constructs that include the S-layer protein as part of a fusionprotein have been reported to express the S-layer protein at theBacillus surface. (See WO-95/19371, describing Bacillus sphaericus),thus enhancing the immunogenicity of the preparation.

Mucosal immunization is already provided against some diseases,including an oral polio vaccine and an oral (drinkable) vaccine againstcholera and diarrhea due to E. coli (an inactivated vaccine). In someembodiments, it is contemplated that the vaccines of the invention maythus comprise an inactivated vaccine.

The present invention contemplates a live vaccine, as such will providea single-dose, long lasting vaccination, because the carrier organism,Helicobacter, will continue to produce the antigen, i.e.,non-Helicobacter pharmacologically active molecule of interest, andboost immunity in vivo. In addition, the vaccines will be administeredin combination with an adjuvant. These adjuvants' comprise moleculessuch as aluminum hydroxide or lipid vesicles that increase the exposuretime for the vaccine by slowing its removal forte site ofadministration. Adjuvants' also act by evoking production ofimmunomodulatory peptides called cytokines and chemokines (Brewer et al.1997, J. Cytokines Cell Mol. Ther., 4:223-246). Thus, the presentvaccines may comprise cytokine adjuvant to enhance immune response.

The transformed Helicobacter or E. coli bacterium, when administeredorally or gastrically to a mammal such as a human or animal, willprovide for the gastro-intestinal colonization, production andpresentation of the desired polypeptide, through the gastric wall, whichis the natural site of colonization. The gastro-intestinal tract issurrounded by an immense immune apparatus specialized in mounting immuneresponse of various types. Gastro-intestinal colonization by recombinantHelicobacter vaccine or peptide producer strain thus enables a muchlonger immune stimulus than traditional vaccination. Additionally,antigen can be presented preferentially to the gut wall or the lumen.

EXAMPLE 12 Helicobacter and Uses thereof as an Appetite Suppressant

The present example is provided to demonstrate the utility of thepresent invention as a method for employing Helicobacter in preparationsand treatment regimens that provide for appetite suppression. Inparticular, delivery to the gut mucosa of a construct that comprisesattenuated Helicobacter together with a non-Helicobacterpharmacologically active molecule of interest that regulates the levelof ghrelin or an agonist of ghrelin, is expected to provide an effectivemeans for providing suppression of the gut-brain axis that regulatesappetite and satiety.

Studies have suggested that ghrelin is an appetite stimulant, i.e.,ghrelin increases food intake in mice (Asakawa et al. 2003, Gut,52(7):947-52). Ghrelin has also been reported to reduce fat utilizationin adipose tissue in rodents (Tschop et al., 2000, Nature, 407: 708-13),as well as to be involved in rat adipogenesis (Choi et al. (2003),Endocrinology, 144 (3):751-9). Ghrelin has also been reported to be ahunger signal, prompting the subject to eat when nutrition availabilityis low.

The teachings of U.S. Pat. No. 6,967,237—Bednarek (2005), relating toghrelin analogs and nucleic acid constructs, and of United States Patentapplication publication 20050191317—Bachmann et al. (2005), relating toghrelin-carrier conjugates, are specifically incorporated herein byreference insofar as these teaching supplement and/or further enhancethe understanding and appreciation of the present invention.

Ghrelin, an endogenous ligand for the growth hormone secretagoguereceptor (GHS-R), stimulates growth hormone (GH) release from culturedpituitary cells in a dose-dependent manner, and is produced and secretedfrom the A-like cells found mainly in the oxyntic glands of the gastricfundus. Ghrelin is now known to play a role in not only GH release, butalso in controlling the appetite and body weight.

Both parenterally and intracerebro-ventricularly administered ghrelinhave been shown to stimulate food intake and increase the body weight ofmice and rats with free access to food, even those animals with GHdeficiency. The control of appetite and body weight may be independentof GH release.

Ghrelin, a 28-amino-acid peptide, is activated when its third serineresidue is acylated by n-octanoic acid, and GHS-R is responsive to thefirst four or five residues including the octanylated serine residue ofthe whole ghrelin peptide. GHS-R has been shown to be present in thepituitary, hypothalamus, adrenal glands, thyroid, pancreas, myocardium,spleen and testes. Ghrelin stimulates the expression of both NPY andAGRP mRNA in the hypothalamus. The central orexigenic effect of ghrelinis mediated by the NPY/AGRP-expressing neurons in the hypothalamus.Ghrelin has also been reported to suppress vagal afferent activity. Theperipheral orexigenic effect of ghrelin may be mediated, at least inpart, by its suppressive effect on the vagal afferent activity. IL-1β isa pro-inflammatory cytokine that mediates the cachectic process bystimulating the expression and release of leptin, and/or by mimickingthe effect on the hypothalamus of excessive negative-feedback signalingfrom leptin.

It is proposed that antagonists to ghrelin if provided to the animal atthe gut mucosa will reduce food intake by an animal and reduce bodyweight gain.

EXAMPLE 13 Cell Wasting Attendant Cancer and AIDS

The present example demonstrates the utility of the present inventionfor use as a preparation that will prevent or inhibit cell wasting,particularly cell wasting associated with diseased states of AIDS andcancer.

Cachexia is a condition characterized by wasting, emaciation, feeblenessand inanition. It was recently reported that the levels of both ghrelinpeptide and ghrelin mRNA in the stomach were up-regulated in a mousemodel of cancer cachexia. In cachectic mice with increased plasma levelsof IL-1β, the plasma concentrations of ghrelin also increased with theprogression of cachexia. This result suggests that a close relationshipmight exist between the ghrelin dynamics and the cachectic processmediated by IL-1. IL-1β is an anorexigenic substance, just like CCK,leptin, gastrin-related protein and bombesin, and antagonizes theactions of ghrelin.

Asakawa et al. reported that parenterally administered IL-1β decreasedNPY mRNA expression in the hypothalamus and preproghrelin mRNAexpression in the stomach, and that intraperitoneally administeredghrelin inhibited the severity of IL-1β-induced anorexia. Helicobacterpylori infection is known to be a major pathogenetic factor in thedevelopment of gastritis, peptic ulcer disease and gastric malignancy.Attachment of H. pylori to the gastric mucosa induces inflammation,which is associated with the release of various cytokines, includingIL-1β.

It has been observed clinically that H. pylori eradication is oftenfollowed by improvement of some nutritional parameters, such as the bodyweight and the serum levels of total cholesterol, total protein andalbumin. H. pylori infection has been reported to be capable ofmodifying the plasma and gastric ghrelin dynamics in Mongolian gerbils.In humans, however, H. pylori infection has been reported not to beassociated with any changes of the plasma ghrelin levels, althougheradication of H. pylori has been shown by some to be associated withincreases of the plasma ghrelin levels.

It is proposed that H. pylori may be used as a carrier to provide amylinto a patient in need thereof, by, for example, acting as a carriervehicle, to the gastric mucosa. In some embodiments, the Helicobactercarrier will be constructed so as to include amylin, amylin agonist,analogs, and derivatives, and amylin agonists (including calcitonins,calcitonin gene-related peptides), and analogs therefore to decreaseghrelin levels.

Amylin antagonists can increase ghrelin levels. Modulation of theeffective levels of amylin, with amylin, amylin agonists, amylinantagonists, or other compounds that decrease the effective level ofamylin such as antibodies, may inhibit, or stimulate in the case ofantagonists and antibodies, ghrelin secretion. Hence, some embodimentsof the method are directed to modulating endogenous levels of ghrelin byincreasing the effective level of amylin or amylin agonists in the body,by direct or indirect means, or by decreasing the effective level ofamylin using amylin antagonists or inhibiting amylin production.

EXAMPLE 14 Treatment of Gauchers Disease

The present example demonstrates the utility of the invention for use asa treatment for a disease resulting from an enzyme deficiency, such asGaucher's disease. Gaucher's disease is the most common lysosomalstorage disorder in humans, and results from a deficiency of the enzyme,glucocerebrosidae (GC). (Nolta et al., (1992), J. Clin. Invest. 90(2):342-348).

Enzyme replacement therapy is provided with a Helicobacter vaccineconstruct that comprises a sequence encoding chemical chaperones.(Sawker et al., (2002), PNAS USA 99(24): 15428-15433). An enhanced levelof functional β-glycosidase (β-Glu, glucocerebrosidase) may thus beobtained. In particular, the chemical chaperone deoxynojirimycin(NN-DNJ) is to be used in the H. pylori construct and administered tothe patient orally or intragastrically.

As part of yet another embodiment of the methods, a Helicobacter-basedconstruct as described herein comprising a vector having anon-Helicobacter pharmacologically active molecule of interest, in thiscase, encoding glucocerebrosidase (GC). Retroviral mediated transfer ofglucocerebrosidase cultured Gaucher bone marrow is described as oneapproach for treating Gauchers disease in Nolta et al. (1992). However,this approach is extremely invasive. Alternative enzyme replacementtherapy employing the Helicobacter-based constructs of the inventionthat include a sequence encoding for the deficient enzyme,glucocerebrosidase, provides a much more attractive and less expensivealternative to such a therapy.

EXAMPLE 15 Treatment of Lymphoma

The present example is presented to demonstrate the utility of thepresent invention to provide a useful preparation that is suitable fortreating and/or inhibiting a bacterial induced malignancy, such aslymphoma, particularly MALT lymphoma, using a vaccination preparationcomprising the Helicobacter vector and/or plasmid vectors as describedherein.

Sutton et al. (2004) (Vaccine, 22 (20): 2541-6) report protectionagainst a bacteria-induced malignancy, specifically primary gastric MALTlymphoma, as a result of vaccination/immunization of an animal againstHelicobacter felis.

Helicobacter pylori constructs of the present disclosure that include avector and/or plasmid vector suitable for providing an immunizingpreparation that includes an immunogenic antigen of interest other thanHelicobacter felis, may also be used to provide vaccination protectionagainst a bacterial-induced malignancy, and in particular, againstprimary gastric MALT lymphoma. By way of example, some embodiments ofthe plasmid vector would include a fusion protein comprising aHelicobacter pylori encoding sequence and a non-Helicobacter pyloriencoding sequence that is, for example, other than a Helicobacter felisantigen species.

Example 16 Live Vaccine Delivery System with H. pylori

The present example demonstrates the utility of using H. pylori in alive vaccine, and in particular the use of H. pylori as part of a liveviral preparation to deliver a protein of interest to the gastric mucosaof an animal. In particular, the present example demonstrates theutility of the present invention for providing delivery of an antigen ofinterest to an animal at the gastric mucosa though the outer surface ofa recombinant Helicobacter pylori outer membrane.

The present studies were primarily done with H. pylori strain B128,variant 7.13 (Franco et al. (2005). The sacB cassette, conferringsucrose sensitivity and kanamycin resistance, was inserted into the hopEgene of H. pylori.

H. pylori is demonstrated herein to be a useful vehicle for vaccinedelivery, and provides an improved bacterial delivery modality for thetreatment of an animal, particularly a human. Factors important inestablishing the utility of the H. pylori based vaccines in thetreatment of humans include the following:

-   1. A majority of persons infected with H. pylori are asymptomatic;-   2. infection with H. pylori induces both adaptive and innate immune    responses;-   3. Infection with H. pylori can persist in the gastric mucosa,    facilitating long-term exposure to antigens;-   4. Infection with H. pylori produces molecules that disrupt the    gastric epithelium, thereby facilitating exposure of bacteria to the    submucosal immune system;-   5. Genome data and molecular techniques are readily available for H.    pylori, thereby facilitating its genetic manipulation.

HopE is an outer membrane protein of H. pylori. The present exampledemonstrates that the nucleic acid sequence encoding this protein can bemodified so as to include a desired sequence encoding a molecule ofinterest (“X”), and H. pylori containing this modified HopE—sequence maythen be used in a vaccine to deliver the molecule of interest (“X”) tothe mucosal surface of an animal.

In the present example, the p60 protein and the HCCA protein are used todemonstrate proof of principle of delivery, and are simple examples ofproteins that may be used to provide such an engineered H. pylori HopEgene.

Methods

Bacterial Strains and Culture

Helicobacter pylori strain B128 (7.13) (52) or 26695 (64) were culturedon Columbia agar (Oxoid, Basingstoke, United Kingdom) supplemented with5% horse blood and incubated under microaerophilic conditions (10% CO2)at 37° C. H. pylori were transformed by natural transformation (65,66).Transformants were selected using chloramphenicol (10 μg/mL) orkanamycin (10 μg/mL). Escherichia coli strain DH5-α (67) was grown inLuriaBertani (LB) medium at 37° C. and transformed by electroporation(68). Transformants were selected using 5% (w/v) sucrose, kanamycin (50ug/mL) or ampicillin (100 ug/mL). Genomic DNA was extracted using theDNeasy Tissue Kit (Quiagen). Plasmid extractions were performed usingthe QIAprep Spin Miniprep Kit (Quiagen).

Construction of Recombinant DNA Molecules

Recombinant DNA molecules were constructed by assembling individual PCRproducts using splicing by overlap extension (SOE) PCR (69). Primerswere designed to introduce overlaps of 12 bp at the termini of PCRproducts, allowing the products to be joined together during PCR (FIG.8). All preparative amplifications were performed using Pwo polymerase(Roche Pharmaceuticals).

Recombinant DNA was constructed to insert a chloramphenicol resistancegene into hopE. A region including a section of, and extending upstream,of hopE was amplified using the primers HopEF3 and HopER3 (Product A).The opa promoter and chloramphenicol resistance gene were amplified frompHe12 (70) using primers CATF and CATR (Product B). A region including asection of, and extending downstream of hopE was amplified using theprimers HopEF4 and HopER5. Products A, B, and C were joined togetherusing primers HopEF3 and HopER5 as described by Chalker and associates(69).

Allelic replacements were achieved using the sacB based selection system(71). The sacB cassette, conferring sucrose sensitivity and kanamycinresistance, was inserted into the gene of interest. Recombinant DNA wastransferred into sucrose-sensitive H. pylori. Transformants were sucroseresistant, resulting from replacement of sacB with transforming DNA.

The sacB cassette was joined to hopE DNA in a number of stages. Firstly,the hopE gene was amplified using the primers HopEF6 and HopER6.Purified amplicon was digested Nco1 and Bgl11 (Roche Pharmaceuticals).Amplicon and similarly digested pBADMyc-HisB (Invitrogen LifeTechnologies) were ligated using the Quick-stick Ligation Kit (Bioline)and transferred to E.coli, producing plasmid pBAD1. Secondly, a regionof DNA, including 804 bp of the intergenic region between hopE and thedownstream putative mraW gene (coding 5-S-adenosylmethyltransferase),was amplified using primers HopEF7 and HopER7. Purified amplicon wasdigested with Bgl11 and Xba11, ligated to similarly digested pBAD1, andtransferred to E. coli to produce plasmid pBAD2. Lastly the sacBcassette was amplified from pENKSF (59) using the primers KanSacF andKanSacR. The amplicon and pBAD2 were digested with Bgl11, ligated andtransferred to E.coli to produce plasmid pBAD3.

Similarly the sacB cassette was inserted into cagA and vacA. For cagA,in the first stage the region directly downstream of cagA was amplifiedusing primers cagAF2 and cagAR2 and digested with Xba1 and Bgl11. In thesecond stage a section of cagA extending to the 3′ end was amplifiedusing primers cagAF1 and cagAR1 and digested with Bgl11 and Nco1. In thefinal stage, the sacB cassette was excised from pBAD3 using BgI11, andinserted into similarly digested pBADMyc-HisB. For vacA, in the firststage a section including the C-terminal region of vacA was amplifiedusing primers vacAF4 and vacAR4, and digested with Hind111 and Xba1. Thefragment was cloned into similarly digested pUC19 to produce pUC1.Secondly, a section of DNA including the N-terminal region of vacA wasamplified using primers vacAF3 and vacAR3, and digested with EcoR1 andKpn1. The fragment was cloned into similarly digested pUC1 to producepUC2. Lastly, the sacB cassette was amplified using the primers KanSacFand KanSacR, and digested with BgI11 to produce pUC3.

Recombinant molecules were constructed to insert DNA coding antigenicsections of the Hepatitis C virus core antigen (HCCA) or L.monocytogenes p60 protein into hopE, replacing the sacB cassette.Recombinant DNA was produced using SOE PCR, joining PCR products A, Band C. A region extending from the start of hopE to nt 573 was amplifiedusing primers HopEF8 and HopER1 (product A). The region of sequencecoding HCCA antigen from aa 7-53 was produced by amplifying primersHCCAF1, HCV.2a, HCV3.s, HCCAR1 (product B). The region downstream ofhopE nt 573 was amplified using HopEF2 and HopER8 (product C). Productresulting form SOE PCR was cloned into plasmid pCR4Blunt-TOPO(Invitrogen Life Technologies) to produce pCR4HCH.

Similarly DNA coding sections of the p60 antigen was inserted into hopEand cagA. For hopE, the region extending from the start of hopE to nt573 was amplified using primers HopEF8 and HopER9 (product A). The p60antigen was produced by annealing primers Lmp60F2 and Lmp60R2 (productB). The region directly downstream of hopE nt 573 was amplified usingprimers HopEF9 and HopER8 (product C). Product resulting form SOE PCRwas cloned into Sma1 digested pUC19 plasmid to produce pUCHLm. For cagA,a region, including the 3′end of cagA was amplified using primers CagAF6and CagER6 (product A). The p60 antigen was produced by annealingprimers Lmp60F2 and Lmp60R2 (product B). The region directly downstreamof cagA was amplified using cagAF6 and cagAR8 (product C). Productresulting from SOE PCR was cloned into Sma1 digested pUC19 to producepUCCLm.

For vacA, a region of vacA was amplified from strain 26695, includingthe gene promoter and signal sequence, using primers vacAF1 and vacAR1(product 1). The p60 antigen was produced by annealing primers Lmp60F3and Lmp60R3 (product B). A section of vacA including the region codingthe autotransporter and mature VacA was amplified using vacAF2 andvacAR2 (product C). Product resulting form SOE PCR digested with XbaIand Kpn1, and cloned into similarly digested pUC2 to produce pUCVLm.

Western Blot Analysis

To determine if recombinant bacteria produced a fusion between HopE andthe antigenic epitope, Western Blot analyses was performed

H. pylori were harvested from a 24 h blood agar into HEPES (pH 7.4) andwere pelleted by centrifugation at 4500 rcf for 5 min and were disruptedby sonication. Lysate was centrifuged at 4500 rcf for 5 min to pelletunlysed bacteria, which were discarded. To harvest the total membranefraction, the supernatant was centrifuged at 100 000 rcf for 60 min. Allprocedures were performed at 4° C. Samples were resuspended in HEPESbuffer and added to an equal volume to Laemmli buffer. Proteins wereseparated in a 12% Tris-HCI Ready Gel (BioRad) by sodium dodecyl sulfate(SDS)-polyaccrylamde gel electrophoresis (PAGE) before electro-transferto 0.45 pM nitrocellulose (Biorad). For the detection of HopE and otherproteins, membranes were blocked with 10% or 5% non-fat dairy milk(NFDM) in Tris buffered saline (TBS) pH 7.4. Membranes were probed withprimary antibodies or secondary antibodies in 1% NFDM in TBS. Polyclonalrabbit anti-HopE antibody was donated (Astra Zenica) and used at adilution of 2:4000. Monoclonal mouse anti-p60 (K3A7) was donated(University of Wurzburg, Germany) and was used at a dilution of 2:4000.Mouse anti-HCCA (C7-50; Sigma) was at a dilution of 2:4000. Secondaryantibodies were conjugated to alkaline phosphatase, and were detectedusing nitro blue tetrazolium chloride/5-Bromo-4-chloro3-indolylphosphatase solution (Roche Applied Science).

Immuno-Based Assays of Surface Localization

H. pylori were harvested from a 24 h blood agar plate into phosphatebuffered saline (PBS; pH 7.4). Bacteria were rinsed a total of 3 timesand standardized to and OD (600 nm) of 0.5. Rinsed bacteria were boundto a poly-L-lysine coated chamber slide overnight at 4° C. Boundbacteria were fixed with 0.25% glutaraldehyde, which was subsequentlyblocked with 100 mM glycine buffer. Between subsequent steps, slideswere rinsed three times in PBS containing 0.05% Tween 20 (PBST). Slideswere locked with 3% bovine serum albumin (BSA) in PBST for 2 h at 37° C.Primary antibodies were diluted 1:200 in PBST containing 1% BSA andincubated for 1.5 h at room temperature. Secondary antibodies conjugatedto Alexafluor 488 were diluted 1:400 in PBST and incubated for 2 h at37° C. Slides were rinsed and surface bound antibodies were detectedusing fluorescence microscopy. Whole cell based ELISA was performedsimilarly to indirect immuno-fluorescence studies with the followingexceptions: bacteria were bound to Maxisorp (Nunc) plates eitherovernight or for 1 hr at 4° C.; primary antibody was diluted 1:300;secondary antibody conjugated to alkaline phosphatase was diluted1:1000; nitrophenyl phosphate (Sigma) was used as a substrate fordetection at 405 nm using a microtiter plate reader.

Results

Construction of Recombinant H. pylori

Recombinant H. pylori B128 (7.13) or 26695 were produced that harboredthe sacB cassette in either the hopE, cagA or vacA gene. The sacBcassette was replaced with DNA coding antigens at specific positionswithin these genes. Both HCCA and p60 antigen DNA were inserted intohopE at a position corresponding to amino acid 168 of mature HopE,within a putative loop structure (51). p60 antigen DNA was also inserteddirectly upstream of the cagA stop codon, corresponding the C-terminalof CagA. Fusions of VacA and antigenic proteins were performed in strainB128 (7.13), which does not produce VacA. p60 antigen DNA was joined tothe 26695 promoter, signal sequence and autotransporter DNA, prior toinsertion into B128 (7.13) vacA.

Analysis of Fusion Proteins and Surface Presentation

To date only HopE fused to HCCA or p60 antigens were analyzed. WesternBlot analysis of H. pylori B128 producing the HCCA (B128:HCCA:hopE) orp60 antigen (B128:p60:hopE) DNA inserted into hopE showed that theantigens were fused to HopE (FIG. 9). Specifically, analyses performedusing the anti-HopE antibody showed that in recombinant H. pylori nativeHopE (about 31 kDa) was replaced with either HopE fused to either p60antigen (about 32 kDa) or HCCA (about 35 kDa). Further analysis usingantigen specific antibodies detected similar sized proteins.

To examine surface presentation, two indirect immuno-based assays ofsurface localization were employed. Immuno fluorescence based microscopyanalysis of H. pylori B128:HCCA:hopE indicated surface presentation ofHopE fused to the HCCA (FIG. 10). However, surface localization of HopEfused to p60 antigen could not be confirmed. These results wereconfirmed by whole cell based ELISA analysis (FIG. 11B). The p60 antigenwas only detected using this method after overnight binding of bacteriato ELISA plates (FIG. 11A).

Studies in this example were done primarily with H. pylori strain B128,variant 7.13 (Franco, 2005)(52). The sacB cassette, conferring sucrosesensitivity and kanamycin resistance, was inserted into the hopE gene ofH. pylori. DNA coding the HCCA or p60 epitope was joined to hopE usingSplicing by Overlapping Extension (SOE) PCR. Recombinant DNA wastransferred into sucrose-sensitive H. pylori using naturaltransformation and homologous recombination. Recombinant H. pylori wassucrose resistant, resulting from replacement of sacB with transformingDNA.

The following oligonucleotide primers presented in Table 1 were used inthe assays described herein (SEQ ID NOS 10-54, respectively in order ofappearance): TABLE 1 Oligonucleotide Primers: Primer Sequence CagAF1AAAACCATGGATTTCAGTAGGGTAGAGCAA cagAF11 ATCAAGAAAGAATTGAATGAGA CagAF2AAAAAGATCTAGGATTAAGGAATACCAAAAACGCA CagAF6 ACCGAA TAAAGGATTAAGGAATACCAAACagAR1 AAAAAGATCTTTAAGATTTTTGGAAACCACCTT CagAR2AAAATCTAGAGGTTATTTTAGGTTGCACGCATTTT CagAR6 AGCTTCAGATTTTTGGAAACCACCTTCagAR8 GTTATTTTAGGTTGCACGCATT CATF TTTTAATCCGCCATATTGTGTTGAA CATRAAGGGTCGTTTAAGGGCACCAATAACT HCCAF1GGTCCTCAGCGGAAGACGAAGCGTAATACAAACAGGAGACCA CA HCCAR1AACATCGCTGGTTTTGCGAGTTGCTCTTACCCCCAAACG HCV2.aCCACCGACTATTTGACCCCCTCCAGGAAATTTAACATCTTGT GGTCTCCT HCV3.sATAGTCGGTGGCGTGTATTTACTACCCAGGCGAGGACCGCGT TTGGGGGT HopEF1AAAAAGATCTAATGGAATTTATGAAAAAGTTTGTAGCT HopEF2ACCAGCGATGTTTGTACCCCTACTTATTGTAACCCTAA HopEF3AATGGAATTTATGAAAAAGTTTGTAGCT HopEF4 TAAACGACCCTTTAAAAGGGTGTCTTTA HopEF6AAAACCATGGATGGAATTTATGAAAAAGTTTGTAGC HopEF7AAAAAGATCTTAAACCCTTTAAAAGGGTGTCTT HopEF8 ATGGAATTTATGAAAAAGTTTGTAGCHopEF9 ACCGAAGATGTTTGTACCCCTACTTATTG HopER1 CCGCTGAGGACCCTTAGCTTCAATGAHopER3 GGCGGATTAAAAAGTGTAGTTATACCCTAAA HopER5 CGGCTTGAAACACCAAAGTCHopER6 AAAAAGATCTAAAAGTGTAGTTATACCCTAAATAA HopER7AAAATCTAGACTTCTGGGCTTGGAGTGATG HopER8 CTTCTGGGCTTGGAGTGATG HopER9AGCTTCAGGACCCTTAGCTTCAATGATT KanSacF AAAAAGATCTCGAACCATTTGAGGTGATAGKanSacR AAAAAGATCTTATAGCCCATTTTCATGCTCTT Lmp60F1GGTCCTGAAGCTGCTAAACCTGCTCCTGCTCCTAGCACCAATCAACAACAAACCGCTCCTAAAGCTCCTACCGAAGATGTT Lmp60F2AAATCTGAAGCTGCTAAACCTGCTCCTGCTCCTAGCACCAATCAACAACAAACCGCTCCTAAAGCTCCTACCGAATAAAGG Lmp60F3CATGCCGAAGCTGCTAAACCTGCTCCTGCTCCTAGCACCAATCAACAACAAACCGCTCCTAAAGCTCCTACCGAACCCGAC Lmp60R1AACATCTTCGGTAGGAGCTTTAGGAGCGGTTTGTTGTTGATTGGTGCTAGGAGCAGGAGCAGGTTTAGCAGCTTCAGGACC Lmp60R2CCTTTATTCGGTAGGAGCTTTAGGAGCGGTTTGTTGTTGATTGGTGCTAGGAGCAGGAGCAGGTTTAGCAGCTTCAGATTT Lmp60R3GTCGGGTTCGGTAGGAGCTTTAGGAGCGGTTTGTTGTTGATTGGTGCTAGGAGCAGGAGCAGGTTTAGCAGCTTCGGCATG vacAF1AAAGGTACCAAAGCCGATAGCATCAGAGAA vacAF2 ACCGAACCCGACAATTACAAGTATCTTATvacAF3 AAAGAATTCAATTTGGTTTCAAGCTCAAATCAGA vacAF4AAATCTAGAACTACATCTGCCACTAATGTGAA vacAR1 AGCTTCGGCATGACTTTGTTGCGGTGTGATvacAR2 AAATCTAGATTAGAAACTATACCTCATTCCTA vacAR3AAAGGTACCGAGCTTGTTGATATTGACTTTGT vacAR4 AAAAAGCTTCATTCTCAGTAGGCGTAGAAT

TABLE 2 Strains of H. pylori used and developed: Strain Antigen DNAInserted Insertion Site Designation B128 (7.13) B128 (7.13) B128 (7.13)p60 hopE B128:p60:hopE B128 (7.13) HCCA hopE B128:HCCA:hopE B128 (7.13)p60 vacA B128:p60:vacA 26695 26659 26695 p60 hopE 26695:p60:hopE 26695HCCA hopE 26695:HCCA:hopE 26695 p60 cagA 26695:p60:cagA

Natural transformation of H. pylori strain B128 yielded recombinantbacteria which produced hopE fused to either p60 or HCCA DNA asconfirmed by sequence analysis. Resulting fusion proteins were largerthan unaltered HopE (FIG. 8). Confirmation of HCCA surface localizationwas shown using fluorescence microscopy (FIG. 9), and was corroboratedby results from whole cell based ELISA analysis.

All documents, patents, journal articles and other materials cited inthe present application are hereby incorporated by reference.

Although the present invention has been fully described in conjunctionwith several embodiments thereof with reference to the accompanyingdrawings, it is to be understood that various changes and modificationsmay be apparent to those skilled in the art. Such changes andmodifications are to be understood as included within the scope of thepresent invention as defined by the appended claims, unless they departtherefrom.

BIBLIOGRAPHY

The references listed below as well as all references cited in theSpecification are incorporated herein by reference to the extent thatthey supplement, explain, provide a background for or teach methodology,techniques and/or compositions employed herein.

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1. A composition comprising a Helicobacter construct having a firstHelicobacter sequence Y1, a second Helicobacter sequence Y2 and anon-Helicobacter sequence X encoding a non-Helicobacter molecule ofinterest, wherein said Helicobacter construct comprises a formula ofFormula 1:


2. The composition of claim 1 wherein the Helicobacter is Helicobacterpylori.
 3. The composition of claim 1 wherein the molecule of interestis heterologous to the Helicobacter pylori species.
 4. The compositionof claim 1 further comprising a promoter sequence.
 5. The composition ofclaim 1 wherein the non-Helicobacter molecule of interest is ghrelin,amylin or an analog thereof.
 6. The composition of claim 2 wherein theHelicobacter construct is further defined as an attenuated Helicobacterpylori.
 7. The composition of claim 1 wherein the molecule of interestcomprises a protein, peptide or nucleic acid molecule.
 8. Thecomposition of claim 1 further defined as comprising a vaccine in apharmacologically acceptable carrier solution.
 9. The composition ofclaim 1 further defined as comprising a plasmid vector having a promotersequence.
 10. A composition comprising a vaccine, wherein said vaccinecomprises a Helicobacter vector plasmid comprising: (a) a Helicobacternucleotide sequence encoding a regulatory sequence capable ofcontrolling the expression of a sequence encoding a non-Helicobacterpharmacologically active molecule of interest; and (b) a sequenceencoding a non-Helicobacter pharmacologically active molecule ofinterest.
 11. The composition of claim 10 further comprising anadjuvant.
 12. The composition of claim 10 further described as suitablefor delivery to a mucosal surface.
 13. The composition of claim 10wherein the non-Helicobacter pharmacologically active molecule ofinterest is a protein, peptide or nucleic acid molecule.
 14. Thecomposition of claim 10 wherein the non-Helicobacter pharmacologicallyactive molecule of interest is ghrelin, amylin or an antagonist oranalog thereof.
 15. A method of immunizing an animal comprising: (a)providing a composition comprising a vaccine according to claim 10 to ananimal; and (b) administering to the animal an effective amount of thecomposition sufficient to elicit an immunologically detectable response.16. The method of claim 15 wherein the immunologically detectableresponse is the production of antibodies.
 17. The method of claim 15wherein the animal is a human.
 18. The method of claim 15 wherein thecomposition is administratered at a mucosal surface of the animal. 19.The method of claim 15 wherein the animal is provided the effectiveamount of the composition comprises a treatment regimen of one or moresequential doses of the composition.
 20. A composition comprising arecombinant cell comprising a sequence encoding a non-Helicobacterpylori pharmacologically active molecule of interest and a Helicobactersequence having a regulatory sequence capable of controlling expressionof the sequence encoding the non-Helicobacter pylori pharmacologicallyactive molecule of interest.
 21. The composition of claim 20 wherein thenucleotide sequence encoding the non-Helicobacter pyloripharmacologically active molecule of interest comprises a secretorysignal polypeptide.
 22. The composition of claim 20 wherein saidrecombinant cell is a Helicobacter.
 23. A pharmaceutical compositioncomprising as an active ingredient the composition of claim 1 in apharmaceutically acceptable dilute, carrier, adjuvant or combinationthereof.
 24. An H. pylori recombinant construct suitable for use in amedicament, said construct comprising: (a) a first H. pylori surfaceantigen encoding nucleic acid sequence; (b) a second H. pylori surfaceantigen encoding nucleic acid sequence; and (c) a nucleic acid sequenceencoding a non-Helicobacter molecule of interest having a first endterminal and a second end terminal, wherein said first H. pylori surfaceantigen encoding nucleic acid sequence is attached to said first endterminal of said non-Helicobacter molecule of interest, and said secondH. pylori surface antigen encoding nucleic acid sequence is attached tosaid second end terminal of said non-Helicobacter molecule of interest.